Team:Goettingen/NoteBook w12
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+ | <span class="date">23rd<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <p class="timeline-title goe-rt">Transformation of Promoter1<sub>rev</sub>/Promoter3<sub>rev</sub> + pSB1C3 , Colony PCR for Transformation of Promoter1<sub>rev</sub>/Promoter3<sub>rev</sub> + pSB1C3, Preparation of new Backup plates from yesterday’s ColonyPCRs/Inoculation...</p> | ||
+ | <div class="timeline-cont"> | ||
+ | |||
+ | <p class="c12-14"><span class="c12-2">Transformation of Promoter1</span><span class="c12-1">rev</span><span class="c12-2">/Promoter3</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>neg. control plates still negative</span></p><p class="c12-9"><span>-</span><span> </span><span>pSB1C3 w/o insert control: 3 big clones on 50 μl-plate, many clones on rest-plate (small, big, medium in size)</span></p><p class="c12-9"><span>-</span><span> </span><span>pSB1C3 + Promoter1</span><span class="c12-10">rev</span><span>: no clones on 50 μl-plate, 10 clones on rest-plate (small or medium size)</span></p><p class="c12-9"><span>-</span><span> </span><span>pSB1C3 + Promoter3</span><span class="c12-10">rev</span><span>: no clones on 50 μl-plate, 6 clones on rest-plate (small or medium size)</span></p><p class="c12-48 c12-14"><span>→ Colony PCR to test for positive clones</span></p><p class="c12-48 c12-14"><span>→ plates stored at 4 °C, big fridge</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Colony PCR for Transformation of Promoter1</span><span class="c12-1">rev</span><span class="c12-2">/Promoter3</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 </span></p><p class="c12-9"><span>-</span><span> </span><span>as described previously (10.7.13)</span></p><p class="c12-9"><span>-</span><span> </span><span>1x RBS</span><span class="c12-10">rev</span><span> in pSB1C3 plasmid as additional control (plasmid prepped on 19.8.13, 211.6 ng/μl → 1:20 dilution → ca. 10 ng/μl), orange tube, termed RBS</span></p><p class="c12-9"><span>-</span><span> </span><span>1x medium size clone from w/o insert control plate picked (purple tube, termed R)</span></p><p class="c12-9"><span>-</span><span> </span><span>10x clones of Promoter1</span><span class="c12-10">rev</span><span> + pSB1C3 transformation picked (termed C1 – C10), blue tubes</span></p><p class="c12-9"><span>-</span><span> </span><span>6x clones of Promoter3</span><span class="c12-10">rev</span><span> + pSB1C3 transformation picked (termed C1 – C6), pink tubes</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of mastermix for 20 reactions, primers: iGEM_38 (VF2) and iGEM_39 (VR)</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of master plate (LB</span><span class="c12-5">Cm</span><span>) → incubation over day at 37 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>25 μl mastermix + 1 μl plasmid dilution or cells</span></p><p class="c12-9"><span>-</span><span> </span><span>PCR protocol: same as on 10.7.13</span></p><p class="c12-9"><span>-</span><span> </span><span>plates stored at 4 °C, big fridge</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Preparation of new Backup plates from yesterday’s ColonyPCRs/Inoculation </span></p><p class="c12-9"><span>-</span><span> </span><span>I wasn’t sure anymore, if I inoculated DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1 – C5, part 6.4 B C1 – C5 and part 6.4A C1 – C8 correctly in the labeled tubes → streak-out of all clones for DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1 – C5, part 6.4 B C1 – C5 and part 6.4A C1 – C8 on new LB</span><span class="c12-5">Cm</span><span> plates</span></p><p class="c12-9"><span>-</span><span> </span><span>in case of DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> and part 6.4 B, the remaining clones on the master plates (that were not inoculated yesterday), were streaked out as well on the new master plate</span></p><p class="c12-9"><span>-</span><span> </span><span>incubation over day at 37 °C, then storage at 4 °C, big fridge</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">MiniPlasmidPreparation of clones inoculated yesterday </span></p><p class="c12-9"><span>-</span><span> </span><span>samples: DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1 – C5, part 6.4 B C1 – C5 and part 6.4A C1 – C8</span></p><p class="c12-9"><span>-</span><span> </span><span>with Nucleospin kit (Macherery-Nagel)</span></p><p class="c12-9"><span>-</span><span> </span><span>1</span><span class="c12-5">st</span><span> elution: 30 μl HPLC water (pre-warmed), incubation for 2 min at 50 °C</span></p><p class="c12-14 c12-56"><span>2</span><span class="c12-5">nd</span><span> elution: 22 μl HPLC water (pre-warmed), incubation for 2 min at 50 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>NanoDrop concentration measurement:</span></p><img src="https://static.igem.org/mediawiki/2013/d/d2/Goe-23.08.13-RT-1.png" /><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Test Restriction Digest of DarR</span><span class="c12-1">rev</span><span class="c12-2">-Term</span><span class="c12-1">rev</span><span class="c12-2"> C1 – C5</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>10 μl in total: 1 μl EcoRI FD + 1 μl PstI FD + 1 μl 10x FD Green buffer + 4 μl dH</span><span class="c12-10">2</span><span>O + 3 μl plasmid mix (ca. 200 – 300 ng plasmid)</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of 6x Mastermix consisting of enzymes, buffer and water</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of 3 μl plasmid-water Mix (plasmids prepped today, see above)</span></p><p class="c12-14 c12-60"><span>DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1 → 3 μl plasmid (88.6 ng/μl)</span></p><p class="c12-14 c12-60"><span>DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C2 → 2 μl plasmid (160.2 ng/μl) + 1 μl dH</span><span class="c12-10">2</span><span>O</span></p><p class="c12-14 c12-60"><span>DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1 → 1 μl plasmid (229.7 ng/μl) + 2 μl dH</span><span class="c12-10">2</span><span>O</span></p><p class="c12-14 c12-60"><span>DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1 → 3 μl plasmid (88.5 ng/μl)</span></p><p class="c12-14 c12-60"><span>DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1 → 2μl plasmid (107.3 ng/μl) + 1 μl dH</span><span class="c12-10">2</span><span>O</span></p><p class="c12-9"><span>-</span><span> </span><span>addition of 7 μl Mastermix</span></p><p class="c12-9"><span>-</span><span> </span><span>incubation at 37 °C for 1 h</span></p><p class="c12-9 c12-37"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Gel run for Test RD of DarR</span><span class="c12-1">rev</span><span class="c12-2">-Term</span><span class="c12-1">rev</span><span class="c12-2"> C1 – C5, plasmids from today’s MiniPrep and Colony PCR for Transformation of Promoter1</span><span class="c12-1">rev</span><span class="c12-2">/Promoter3</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 </span></p><p class="c12-9"><span>-</span><span> </span><span>3x 1%-agarose-1xTAE gel</span></p><p class="c12-9"><span>-</span><span> </span><span>addition of 5 μl 5xLD to 25 μl Colony PCR reaction and loading of 5 μl PCR reaction supplied with 5xLD (during loading, some sample Colony PCR Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C4 was spilled over the well…)</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 1 μl uncut DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1 – C5 plasmid + 1 μl 5x LD + 3 μl dH</span><span class="c12-10">2</span><span>O; loading of 5 μl RD reaction mix</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 1 μl uncut part 6.4 B C1 – C5 and part 6.4A C1 – C8 plasmid + 1 μl 5x LD + 3 μl dH</span><span class="c12-10">2</span><span>O</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 3 μl 2 log ladder as a marker</span></p><p class="c12-9"><span>-</span><span> </span><span>run at 100 V</span></p><p class="c12-9"><span>-</span><span> </span><span>EtBr staining + destaining in water</span></p><p class="c12-9"><span>-</span><span> </span><span>UV detection</span></p><p class="c12-14"><span> </span></p><p class="c12-14"><span class="c12-2">Gel 1:</span></p><p class="c12-14"><span>Loading: Marker/uncut DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1/ RD DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1/ uncut DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C2/ RD DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C2/ uncut DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C3/ RD DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C3/ uncut DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C4/ RD DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C4/ uncut DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C5/ RD DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C5/part 6.4 B C1 plasmid/part 6.4 B C2 plasmid/part 6.4 B C3 plasmid/part 6.4 B C4 plasmid/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/e/ee/Goe-23.08.13-RT-2.png" /><p class="c12-8"><span></span></p><p class="c12-14"><span>expected sizes for DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> plasmids: 630 bp (DarR</span><span class="c12-10">rev</span><span>) + 130 bp (Terminator</span><span class="c12-10">rev</span><span>) = 760 bp and 2070 bp from pSB1C3 backbone </span></p><p class="c12-19 c12-14 c12-63"><span>→ all DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> clones are positive → sequencing of C2 and C3 by SeqLab (because these clones have highest plasmid concentration)</span></p><p class="c12-14 c12-48"><span>→ all purified part 6.4 B plasmids look normal except for plasmid part 6.4 B C1 and C3 → sequencing of C2 and C5 by SeqLab (because these clones have highest plasmid concentration and because their plasmids seem to be ok)</span></p><p class="c12-14"><span> </span></p><p class="c12-14"><span class="c12-2">Gel 2:</span></p><p class="c12-14"><span>Loading: Marker/part 6.4 B C5 plasmid/ part 6.4A C1 plasmid/ part 6.4A C2 plasmid/ part 6.4A C3 plasmid/ part 6.4A C4 plasmid/ part 6.4A C5 plasmid/ part 6.4A C6 plasmid/ part 6.4A C7 plasmid/ part 6.4A C8 plasmid/Colony PCR RBS</span><span class="c12-10">rev</span><span>+pSB1C3 plasmid/ Colony PCR Re-ligand/Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C1/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C2/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C3/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/3/30/Goe-23.08.13-RT-3.png" /><p class="c12-8"><span></span></p><p class="c12-14"><span>expected sizes for Promoter1</span><span class="c12-10">rev</span><span> and Promoter3</span><span class="c12-10">rev</span><span> Colony PCR products: 310 bp (VR, VF2) + 112 bp Promoter1/3</span><span class="c12-10">rev</span><span> = 422 bp</span></p><p class="c12-14"><span>expected sizes for RBS</span><span class="c12-10">rev</span><span>+pSB1C3 and Re-ligand: 310 bp (VR, VF2) + 12 bp (RBS</span><span class="c12-10">rev</span><span>) = 322 bp </span></p><p class="c12-15 c12-14"><span>→ </span><span>all purified part 6.4 B/A plasmids look normal → sequencing of part 6.4 A C2, C3, C5 and C8 by SeqLab (because these clones have highest plasmid concentration) </span></p><p class="c12-15 c12-14"><span>→ </span><span>Colony PCR: for RBS</span><span class="c12-10">rev</span><span>+pSB1C3 and Re-ligand, the expected band was observed, band at same bp for Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C1 – C3 (negative clones) </span></p><p class="c12-14 c12-64"><span> </span></p><p class="c12-14"><span class="c12-2">Gel3:</span></p><p class="c12-14"><span>Loading: Marker/ Colony PCR RBS</span><span class="c12-10">rev</span><span>+pSB1C3 plasmid/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C4/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C5/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C6/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C7/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C8/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C9/ Colony PCR Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C10/ Colony PCR Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C1/ Colony PCR Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C2/ Colony PCR Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C3/ Colony PCR Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C4/ Colony PCR Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C5/ Colony PCR Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C6/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/c/ce/Goe-23.08.13-RT-4.png"/><p class="c12-8"><span></span></p><p class="c12-14"><span>expected sizes for Promoter1</span><span class="c12-10">rev</span><span> and Promoter3</span><span class="c12-10">rev</span><span> Colony PCR products: 310 bp (VR, VF2) + 112 bp Promoter1/3</span><span class="c12-10">rev</span><span> = 422 bp</span></p><p class="c12-14"><span>expected sizes for RBS</span><span class="c12-10">rev</span><span>+pSB1C3 and Re-ligand: 310 bp (VR, VF2) + 12 bp (RBS</span><span class="c12-10">rev</span><span>) = 322 bp </span></p><p class="c12-15 c12-14"><span>→ </span><span> </span><span>Colony PCR: for RBS</span><span class="c12-10">rev</span><span>+pSB1C3, the expected band was observed, band at same bp for Promoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C5 and for Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C1 – C4 and C6 (negative clones); band at ca. 400 bp for romoter1</span><span class="c12-10">rev</span><span>+pSB1C3 C4 and Promoter3</span><span class="c12-10">rev</span><span>+pSB1C3 C5 (= positive clones)→ on Monday: inoculation of these clones for MiniPrep, test RD and sequencing</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Preparations of samples for sequencing by SeqLab </span></p><p class="c12-14"><span>9 – DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C2 + VF2</span></p><p class="c12-14"><span>10 – DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C2 + VR</span></p><p class="c12-14"><span>11 – DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C3 + VF2</span></p><p class="c12-14"><span>12 - DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C3 + VR</span></p><p class="c12-14"><span>13 – part6.4 B C2 + VF2</span></p><p class="c12-14"><span>14 – part 6.4 B C5 + VF2</span></p><p class="c12-14"><span>15 – part 6.4 A C2 + VF2</span></p><p class="c12-14"><span>16 – part 6.4 A C3 + VF2</span></p><p class="c12-14"><span>17- part 6.4 A C5 + VF2</span></p><p class="c12-14"><span>18 – part 6.4 A C8 + VF2</span></p><p class="c12-14"><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>for all samples: 6 μl plasmid + 1 μl iGEM_38 1:5 or iGEM_39 1:5</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Cryostocks and Minipreps of Ribo A C4 and C5 inocculated by Nina yesterday </span></p><p class="c12-14"><span>Following clones were frozen away prepped:</span><span class="c12-2"> </span></p><a href="#" name="a1b43580d341d5ffaa2be4918c201dba4788724f"></a><a href="#" name="16"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-29"><p class="c12-7"><span class="c12-2">Samples</span></p></td><td class="c12-29"><p class="c12-7"><span class="c12-2">Procedures done</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C4 3</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock and Mini</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C4 4</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock and Mini</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C4 6</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock and Mini</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C4 7</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C4 8</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock and Mini</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C5 2</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock and Mini</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C5 3</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock and Mini</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C5 6</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock and Mini</span></p></td></tr><tr><td class="c12-29"><p class="c12-7"><span>C5 8</span></p></td><td class="c12-29"><p class="c12-7"><span>Cryostock and Mini</span></p></td></tr></tbody></table><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span>- C4 7 was dropped during the miniprep</span></p><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2">Nanodrop: </span></p><p class="c12-14"><span>All ca. 400-500ng/µl , good ratio, exact measurements on the vials</span></p><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2">Sequencing by SeqLab: </span></p><p class="c12-14"><span>1: Ribo A C4 6 VF</span></p><p class="c12-14"><span>2: Ribo A C4 6 VR</span></p><p class="c12-14"><span>3: Ribo A C4 8 VF</span></p><p class="c12-14"><span>4: Ribo A C4 8 VR</span></p><p class="c12-14"><span>5: Ribo A C5 6 VF</span></p><p class="c12-14"><span>6: Ribo A C5 6 VR</span></p><p class="c12-14"><span>7: Ribo A C5 8 VF</span></p><p class="c12-14"><span>8: Ribo A C5 8 VR</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Inoculation of 10ml cultures of Ribo A C4 7 and 8, the DAC cells and an empty vector as control (names to be added later) for an experiment with plates containing either the reporter system or the DAC producing cells in the agar. Clones were inoculated by Katrin G. yesterday. </span></p><p class="c12-14"><span>Our Ribo A clones didn´t grow at all, the other two cultures were harvested at OD600=2, washed and frozen away at -80°C in Glycerin as follows: </span></p><p class="c12-14"><span>→ harvest 4x 2ml of the culture in 2ml eppis</span></p><p class="c12-14"><span>→ centrifuge down</span></p><p class="c12-14"><span>→ discard supernatant</span></p><p class="c12-14"><span>→ resuspend cells in 100µl LB medium</span></p><p class="c12-14"><span>→ transfer the resuspended cells into a 2ml screw-cap-eppi</span></p><p class="c12-14"><span>→ add 200µl Glycerin</span></p><p class="c12-14"><span>→ Vortex and freeze at -80°C</span></p><p class="c12-8"><span></span></p> | ||
+ | <br/> | ||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | <p class="timeline-title goe-dac">SDS Page of | ||
+ | dialysis and elution samples</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt">SDS Page of | ||
+ | dialysis and elution samples</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Mix 15µl of samples with 5µl Pab | ||
+ | and 5µl buffer W</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation for 10min at 93°C </span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Mix each sample with 4µl protein marker</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Load whole volume on 15% SDS-gel</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Run for 5min at 90V and 1h at 120V</span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt"> </span></b></p> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="tlob" id="tl_0822"> | ||
+ | <span class="date" style="left:-5px">22nd<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont" style="left:-10px"> | ||
+ | <p class="timeline-title goe-rt">Colony PCR for DarR<sub>rev</sub>-Term<sub>rev</sub> transformation , Colony PCR for part6.3 B + RBS<sub>rev</sub> transformation, Gel run for Colony PCR for DarR<sub>rev</sub>-Term<sub>rev</sub> transformation, Sequencing of part6.3 B C1, C2 and C3 plasmids by G2L, Plates of part6.3 A + RBS<sub>rev</sub> transformation...</p> | ||
+ | <div class="timeline-cont"> | ||
+ | |||
+ | <p class="c12-14"><span class="c12-2">Transformation from 21.8.13 </span></p><p class="c12-9"><span>-</span><span> </span><span>neg. control: no clones on 50 μl-plate, no clones on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>Term</span><span class="c12-10">rev</span><span> vector w/o insert: no clones on 50 μl-plate, few clones on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>Term</span><span class="c12-10">rev</span><span> vector + DarR</span><span class="c12-10">rev</span><span> insert: few clones on 50 μl-plate, many clones on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>part 6.3 A vector w/o insert: no clones on 50 μl-plate, 1 clone on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>part 6.3 A vector + RBS</span><span class="c12-10">rev</span><span> insert: no clones on 50 μl-plate, few clones on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>part 6.3 B vector w/o insert: no clones on 50 μl-plate, no clones on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>part 6.3 B vector + RBS</span><span class="c12-10">rev</span><span> insert: no clones on 50 μl-plate, some clones on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>pSB1C3 vector w/o insert: 1 or 2 clones on 50 μl-plate, several clones on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>pSB1C3 vector + Promoter1</span><span class="c12-10">rev</span><span> insert: no clones on 50 μl-plate, no clones on rest-plate</span></p><p class="c12-9"><span>-</span><span> </span><span>pSB1C3 vector + Promoter3</span><span class="c12-10">rev</span><span> insert: no clones on 50 μl-plate, few clones on rest-plate </span></p><p class="c12-15 c12-14"><span>→ colony PCR for DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> transformation</span></p><p class="c12-15 c12-14"><span>→ further incubation of all other plates, until clones are bigger/grown</span></p><p class="c12-15 c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Colony PCR for DarR</span><span class="c12-1">rev</span><span class="c12-2">-Term</span><span class="c12-1">rev</span><span class="c12-2"> transformation</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>as described previously (10.7.13)</span></p><p class="c12-9"><span>-</span><span> </span><span>1x Term</span><span class="c12-10">rev</span><span> C1 plasmid as additional control (plasmid prepped on 16.8.13, 102.8 ng/μl → 1:10 dilution → ca. 10 ng/μl), blue tube (termed T)</span></p><p class="c12-9"><span>-</span><span> </span><span>1x Re-ligand (termed R), pink tube</span></p><p class="c12-9"><span>-</span><span> </span><span>12x DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> clones (termed C1 – C12), purple tubes</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of mastermix for 15 reactions, primers: iGEM_38 (VF2) and iGEM_39 (VR)</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of master plate (LB</span><span class="c12-5">Cm</span><span>) → incubation over day at 37 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>25 μl mastermix + 1 μl plasmid dilution or cells</span></p><p class="c12-9"><span>-</span><span> </span><span>PCR protocol: same as on 10.7.13</span></p><p class="c12-9"><span>-</span><span> </span><span>plates stored at 4 °C, big fridge</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Plates for part 1 – 4 C1 – C3</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>plates taken out, since all clones grew</span></p><p class="c12-9"><span>-</span><span> </span><span>plates put to 4 °C, big fridge</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Colony PCR for part6.3 B + RBS</span><span class="c12-1">rev</span><span class="c12-2"> transformation</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>after some additional incubation time</span></p><p class="c12-9"><span>-</span><span> </span><span>as described previously (10.7.13)</span></p><p class="c12-9"><span>-</span><span> </span><span>1x part6.3 B C2 plasmid as additional control (plasmid prepped on 19.8.13, 220.3 ng/μl → 1:20 dilution → ca. 11 ng/μl), purple tube, termed 6.3 B</span></p><p class="c12-9"><span>-</span><span> </span><span>neg. control plates still without colonies</span></p><p class="c12-9"><span>-</span><span> </span><span>still no religands → performing dephosphorylation longer than actually necessary seems to be helpful (see 20.8.13)</span></p><p class="c12-9"><span>-</span><span> </span><span>1 or 2 clones on 50 μl plate, many clones on rest-plate: 13x clones picked (termed C1 – C13), green tubes</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of mastermix for 15 reactions, primers: iGEM_38 (VF2) and iGEM_39 (VR)</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of master plate (LB</span><span class="c12-5">Cm</span><span>) → incubation over day at 37 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>25 μl mastermix + 1 μl plasmid dilution or cells</span></p><p class="c12-9"><span>-</span><span> </span><span>PCR protocol: same as on 10.7.13</span></p><p class="c12-9"><span>-</span><span> </span><span>plates stored at 4 °C, big fridge </span></p><p class="c12-15 c12-14"><span>→ 12 bp (size of RBS</span><span class="c12-10">rev</span><span>) difference between positive and negative clones, one would never see in a gel… PCR thrown away…</span></p><p class="c12-15 c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Gel run for Colony PCR for DarR</span><span class="c12-1">rev</span><span class="c12-2">-Term</span><span class="c12-1">rev</span><span class="c12-2"> transformation </span></p><p class="c12-9"><span>-</span><span> </span><span>1%-agarose-1xTAE gel</span></p><p class="c12-9"><span>-</span><span> </span><span>addition of 5 μl 5xLD to 25 μl PCR reaction</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 3 μl 2 log ladder as a marker</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 5 μl PCR reaction supplied with 5xLD</span></p><p class="c12-9"><span>-</span><span> </span><span>run at 100 V</span></p><p class="c12-9"><span>-</span><span> </span><span>EtBr staining + destaining in water</span></p><p class="c12-9"><span>-</span><span> </span><span>UV detection</span></p><p class="c12-14"><span> </span></p><p class="c12-14"><span>Loading: Marker/Term</span><span class="c12-10">rev</span><span> plasmid/Re-ligand/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C1/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C2/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C3/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C4/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C5/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C6/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C7/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C8/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C9/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C10/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C11/ DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> C12/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/6/6f/Goe-22.08.13-RT-1.png" /><p class="c12-8"><span></span></p><p class="c12-9"><span>-</span><span> </span><span>expected product sizes</span></p><p class="c12-14 c12-19"><span>negative clone: 310 bp (VR, VF2) + 130 bp (Terminator</span><span class="c12-10">rev</span><span>) = 440 bp</span></p><p class="c12-19 c12-14"><span>positive clone: 310 bp (VR, VF2) + 130 bp (Terminator</span><span class="c12-10">rev</span><span>) + 630 bp (DarR</span><span class="c12-10">rev</span><span>) = 1070 bp </span></p><p class="c12-14 c12-15"><span>→ Religand plasmid corresponds to Term</span><span class="c12-10">rev</span><span> plasmid, as expected</span></p><p class="c12-15 c12-14"><span>→ all DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> clones appear to be positive → inoculate 3 clones for MiniPrep tomorrow</span></p><p class="c12-9"><span>-</span><span> </span><span>PCR reactions stored at 4 °C, small fridge</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Sequencing of part6.3 B C1, C2 and C3 plasmids by G2L </span></p><p class="c12-14"><span>sequencing by SeqLab failed; for both clones, sequence stopped at same region: possible reason: secondary structures formed during sequencing </span></p><p class="c12-14"><span>5 μl total reaction: ca. 300 ng plasmid + 10 % DMSO (to prevent secondary structures) + 1 μl primer iGEM_38, 1:20 diluted</span></p><p class="c12-14"><span> </span></p><p class="c12-14"><span>Preparation of the following samples</span></p><p class="c12-9"><span>-</span><span> </span><span>ppra_1: 2 μl plasmid prepped on 19.8.13 (268.4 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water</span></p><p class="c12-9"><span>-</span><span> </span><span>ppra_2: 2 μl plasmid prepped on 19.8.13 (220.3 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water</span></p><p class="c12-9"><span>-</span><span> </span><span>ppra_3: 2 μl plasmid prepped on 19.8.13 (247.2 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water</span></p><p class="c12-9"><span>-</span><span> </span><span>ppra_4: 1 μl plasmid prepped on 21.8.13 (305.4 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water</span></p><p class="c12-9"><span>-</span><span> </span><span>ppra_5: 1 μl plasmid prepped on 21.8.13 (332.1 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water</span></p><p class="c12-9"><span>-</span><span> </span><span>ppra_6: 1 μl plasmid prepped on 21.8.13 (328.8 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water </span></p><p class="c12-14"><span>prior to sequencing, the samples have to be denatured → remark was entered into excel-sheet</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Plates of part6.3 A + RBS</span><span class="c12-1">rev</span><span class="c12-2"> transformation </span></p><p class="c12-9"><span>-</span><span> </span><span>still no clones on neg. control</span></p><p class="c12-9"><span>-</span><span> </span><span>3 clones on w/o insert plate</span></p><p class="c12-9"><span>-</span><span> </span><span>8 clones on w/ insert plate</span></p><p class="c12-9 c12-37"><span></span></p><p class="c12-14"><span class="c12-2">Inoculation of DarR</span><span class="c12-1">rev</span><span class="c12-2">-Term</span><span class="c12-1">rev</span><span class="c12-2"> clones, part6.3 B + RBS</span><span class="c12-1">rev</span><span class="c12-2">(= part 6.4 B) and part 6.3 A + RBS</span><span class="c12-1">rev</span><span class="c12-2">(= part 6.4 A) clones for MiniPrep </span></p><p class="c12-9"><span>-</span><span> </span><span>inoculation of</span></p><p class="c12-19 c12-14"><span>DarR</span><span class="c12-10">rev</span><span>-Term</span><span class="c12-10">rev</span><span> clones C1 – C5→ Test restriction digest</span></p><p class="c12-19 c12-14"><span> B clones C1 – C5 (no re-ligands)→ sequencing</span></p><p class="c12-19 c12-14"><span>part6.4 A clones C1 – C8 (3 re-ligands and 8 clones on WITH-insert plate→ sequencing</span></p><p class="c12-9"><span>-</span><span> </span><span>in 4 ml LB</span><span class="c12-5">Cm</span></p><p class="c12-9"><span>-</span><span> </span><span>incubation at 37 °C ON</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Run the colony PCR from yesterday on a gel </span></p><p class="c12-14"><span>Gel doc: </span></p><p class="c12-14"><span>Wells: M/Ribo A C4 clone 1/2/3/4/5/6/7/8/9/10/blank/religant from control plate w/o insert</span></p><img src="https://static.igem.org/mediawiki/2013/6/6a/Goe-22.08.13-RT-2.png" /><p class="c12-8"><span></span></p><p class="c12-14"><span>Wells: M/Ribo A C5 clone 1/2/3/4/5/6/7/8/9/10/blank/religant from control plate w/o insert</span></p><img src="https://static.igem.org/mediawiki/2013/7/78/Goe-22.08.13-RT-3.png" /><p class="c12-8"><span></span></p><p class="c12-15 c12-14"><span>→ all clones with the high band should contain the Ribo A insert</span></p><p class="c12-15 c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2">Following clones were choosen to prepare a miniprep: </span></p><p class="c12-14"><span class="c12-2">Ribo A C4 clones 3/4/6/7/8 (all others didn’t grew or showed wrong bands)</span></p><p class="c12-14"><span class="c12-2">Ribo A C5 clones 2/3/6/8 (all others didn’t grew or showed wrong bands) </span></p><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2">Miniprep of the DAC team Lm cryostock colony </span></p><p class="c12-14"><span>Nanodrop:</span></p><a href="#" name="7bbf5b25cd87b2c987fccd03868110c664ff6170"></a><a href="#" name="14"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-17"><p class="c12-7"><span>Sample</span></p></td><td class="c12-46"><p class="c12-7"><span>ng/µl</span></p></td><td class="c12-25"><p class="c12-7"><span>A</span><span class="c12-10">260nm</span><span>/A</span><span class="c12-10">280nm</span></p></td><td class="c12-25"><p class="c12-7"><span>A</span><span class="c12-10">260nm</span><span>/A</span><span class="c12-10">230nm</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C4 3</span></p></td><td class="c12-46"><p class="c12-7"><span>15.8</span></p></td><td class="c12-25"><p class="c12-7"><span>0.82</span></p></td><td class="c12-25"><p class="c12-7"><span>0.18</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C4 4</span></p></td><td class="c12-46"><p class="c12-7"><span>105.8</span></p></td><td class="c12-25"><p class="c12-7"><span>1.92</span></p></td><td class="c12-25"><p class="c12-7"><span>0.89</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C4 6</span></p></td><td class="c12-46"><p class="c12-7"><span>46.8</span></p></td><td class="c12-25"><p class="c12-7"><span>1.44</span></p></td><td class="c12-25"><p class="c12-7"><span>0.41</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C4 7</span></p></td><td class="c12-46"><p class="c12-7"><span>223.6</span></p></td><td class="c12-25"><p class="c12-7"><span>1.69</span></p></td><td class="c12-25"><p class="c12-7"><span>0.93</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C4 8</span></p></td><td class="c12-46"><p class="c12-7"><span>85.2</span></p></td><td class="c12-25"><p class="c12-7"><span>1.53</span></p></td><td class="c12-25"><p class="c12-7"><span>0.73</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C5 2</span></p></td><td class="c12-46"><p class="c12-7"><span>23.3</span></p></td><td class="c12-25"><p class="c12-7"><span>1.74</span></p></td><td class="c12-25"><p class="c12-7"><span>2.27</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C5 3</span></p></td><td class="c12-46"><p class="c12-7"><span>56.0</span></p></td><td class="c12-25"><p class="c12-7"><span>1.97</span></p></td><td class="c12-25"><p class="c12-7"><span>0.87</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C5 6</span></p></td><td class="c12-46"><p class="c12-7"><span>154.9</span></p></td><td class="c12-25"><p class="c12-7"><span>1.56</span></p></td><td class="c12-25"><p class="c12-7"><span>0.71</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>C5 8</span></p></td><td class="c12-46"><p class="c12-7"><span>-0.1</span></p></td><td class="c12-25"><p class="c12-7"><span>-0.06</span></p></td><td class="c12-25"><p class="c12-7"><span>-0.05</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>DAC Lm</span></p></td><td class="c12-46"><p class="c12-7"><span>36.5</span></p></td><td class="c12-25"><p class="c12-7"><span>1.75</span></p></td><td class="c12-25"><p class="c12-7"><span>1.91</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>Second elute C5 2</span></p></td><td class="c12-46"><p class="c12-7"><span>17.5</span></p></td><td class="c12-25"><p class="c12-7"><span>1.46</span></p></td><td class="c12-25"><p class="c12-7"><span>0.65</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>Second elute DAC Lm</span></p></td><td class="c12-46"><p class="c12-7"><span>51.1</span></p></td><td class="c12-25"><p class="c12-7"><span>1.83</span></p></td><td class="c12-25"><p class="c12-7"><span>2.41</span></p></td></tr><tr><td class="c12-17"><p class="c12-7"><span>Second measurement C5 8</span></p></td><td class="c12-46"><p class="c12-7"><span>3.2</span></p></td><td class="c12-25"><p class="c12-7"><span>1.37</span></p></td><td class="c12-25"><p class="c12-7"><span>0.37</span></p></td></tr></tbody></table><p class="c12-14"><span>Stored in to do box</span></p><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2">Following plasmids were choosen for sequencing: </span></p><a href="#" name="a00bec62ab28da8e7bf5f8e4e33a2bc5bcc17a92"></a><a href="#" name="15"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-50"><p class="c12-7"><span>Seq number</span></p></td><td class="c12-28"><p class="c12-7"><span>Plasmid + primer</span></p></td></tr><tr><td class="c12-50"><p class="c12-7"><span>1</span></p></td><td class="c12-28"><p class="c12-7"><span>C4 clone 7 for</span></p></td></tr><tr><td class="c12-50"><p class="c12-7"><span>2</span></p></td><td class="c12-28"><p class="c12-7"><span>C4 clone 7 rev</span></p></td></tr><tr><td class="c12-50"><p class="c12-7"><span>3</span></p></td><td class="c12-28"><p class="c12-7"><span>C4 clone 6 for</span></p></td></tr><tr><td class="c12-50"><p class="c12-7"><span>4</span></p></td><td class="c12-28"><p class="c12-7"><span>C4 clone 6 rev</span></p></td></tr><tr><td class="c12-50"><p class="c12-7"><span>5</span></p></td><td class="c12-28"><p class="c12-7"><span>C5 clone 3 for</span></p></td></tr><tr><td class="c12-50"><p class="c12-7"><span>6</span></p></td><td class="c12-28"><p class="c12-7"><span>C5 clone 3 rev</span></p></td></tr><tr><td class="c12-50"><p class="c12-7"><span>7</span></p></td><td class="c12-28"><p class="c12-7"><span>C5 clone 6 for</span></p></td></tr><tr><td class="c12-50"><p class="c12-7"><span>8</span></p></td><td class="c12-28"><p class="c12-7"><span>C5 clone 6 rev</span></p></td></tr></tbody></table><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span>Again inocculation of all preped Ribo A C4 and C5 clones that were already prepped, for preparation of Cryostocks and a new miniprep, as the old one showed smears</span></p><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2">Katrin Gunka</span></p><p class="c12-14"><span>Preparation of plates with Lb medium also containing the DAC team Lm stain. One plate just like this, another one also containing </span></p><p class="c12-8"><span></span></p> | ||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | <p class="timeline-title goe-array">RT-PCR</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family: | ||
+ | "Arial","sans-serif";text-decoration:none">RT-PCR</span></h2> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">Another | ||
+ | gel to check the digestion from the day before and to check the newly ordered | ||
+ | primers for ydaO and ktrA.</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">The | ||
+ | gel looked fine, so the RT-PCR can get started</span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-GB" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif"">Concentrations digested RNA</span></b></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">digestion | ||
+ | 20.08.13</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent: | ||
+ | -18.0pt"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">wt: 76 ng/</span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">µl</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent: | ||
+ | -18.0pt"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">44: 73 ng/µl</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent: | ||
+ | -18.0pt"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">46: 83 ng/µl</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent: | ||
+ | -18.0pt"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">wt*: 87 ng/</span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">µl</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent: | ||
+ | -18.0pt"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">44*: 73 ng/µl</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent: | ||
+ | -18.0pt"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">46*: 69 ng/µl</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">digestion | ||
+ | 21.08.13</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent: | ||
+ | -18.0pt"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">wt: 63 ng/</span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">µl</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style="margin-left:72.0pt;text-indent: | ||
+ | -18.0pt"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">44: 64,5 ng/µl</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style="margin-left:72.0pt;"><span lang="EN-GB" style="font-family:"Courier New"">o<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">46: | ||
+ | 72 ng/µl</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""><img width="337" height="300" id="Bild 18" src="https://static.igem.org/mediawiki/2013/2/25/Goe-arr-Image008.jpg" alt="Macintosh HD:Users:jangundlach:Desktop:130821_progress:Folie6.jpg"></span></p> | ||
+ | <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family: | ||
+ | "Arial","sans-serif";text-decoration:none">Results RT-PCR</span></h2> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""><img width="465" height="363" id="Bild 9" src="https://static.igem.org/mediawiki/2013/8/86/Goe-arr-Image009.jpg" alt="Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130903_ Ergebnisse Zusammenfassung:Folie1.jpg"></span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""><img width="454" height="347" id="Bild 10" src="https://static.igem.org/mediawiki/2013/9/94/Goe-arr-Image010.jpg" alt="Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130903_ Ergebnisse Zusammenfassung:Folie2.jpg"></span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">no | ||
+ | significant differences. All genes seem to be downregulated in both strains | ||
+ | (1344 = much c-di-AM; 1346 = little c-di-AMP)</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Wingdings">Ø<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">for | ||
+ | better differences a new RT-PCR with differrent conditions are tested | ||
+ | (medium: low and high phosphate medium)</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"; | ||
+ | color:red">These results show us, that there is no real difference between | ||
+ | the strains, because all genes are downregulated.<i>ptsH, hrcA and groEL, </i>served | ||
+ | as control. We are mainly interested in <i>ydaO </i>and</span><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"; | ||
+ | color:red"> </span><i><span lang="EN-GB" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif";color:red">ktrA</span></i><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif";color:red">. <i>YdaO | ||
+ | </i>is the gene behind the riboswitch, <i>ktrA</i> is a phosphate channel. | ||
+ | Due to the fact that there is no difference between 1344 and 1346 compared to | ||
+ | the wildtype in CSE 0,5% Glucose medium, we did the exact same setup only in | ||
+ | low</span><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"; | ||
+ | color:red"> & </span><span lang="EN-GB" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif";color:red">high phosphate medium (see below).</span></p> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="tlob" id="tl_0821"> | ||
+ | <span class="date">21st<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <p class="timeline-title goe-rt">Plasmid Mini Prep, | ||
+ | Transformation of ligations prepared yesterday, observation of clones of Ribo A C4 and C5 CFP under UV light...</p> | ||
+ | <div class="timeline-cont"> | ||
+ | |||
+ | <p class="c12-14"><span class="c12-2">CryoStocks</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of DMSO cryostocks of all inoculated clones (according to protocol described previously)</span></p><p class="c12-8"><span class="c12-2"></span></p><p class="c12-14"><span class="c12-2">Plasmid Mini Prep </span></p><p class="c12-9"><span>-</span><span> </span><span>MiniPrep was done using the culture volume that remained after preparing the cryostocks (see above)</span></p><p class="c12-9"><span>-</span><span> </span><span>kit: Nucleospin, Macherey-Nagel</span></p><p class="c12-9"><span>-</span><span> </span><span>cells were harvested by centrifuging at 13000 g, 1 min</span></p><p class="c12-9"><span>-</span><span> </span><span>elution: 1x 30 μl HPLC water (pre-warmed), incubation for 3 min at 50 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>NanoDrop concentration measurement:</span></p><img src="https://static.igem.org/mediawiki/2013/6/6c/Goe-21.08.13-RT-1.png" /><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Transformation of ligations prepared yesterday </span></p><p class="c12-9"><span>-</span><span> </span><span>according to protocol in methods folder</span></p><p class="c12-9"><span>-</span><span> </span><span>with XL1-Blue comp. cells</span></p><p class="c12-9"><span>-</span><span> </span><span>neg. control was done</span></p><p class="c12-9"><span>-</span><span> </span><span>when adding the Promoter1</span><span class="c12-10">rev</span><span> + pSB1C3 ligation mix to the comp. cells, a drop of the comp. cells was spilled…</span></p><p class="c12-9"><span>-</span><span> </span><span>500 μl were removed after centrifuging</span></p><p class="c12-9"><span>-</span><span> </span><span>plating on LB</span><span class="c12-5">Cm</span><span> plates</span></p><p class="c12-9"><span>-</span><span> </span><span>incubation at 37 °C</span></p><p class="c12-8 c12-40"><span></span></p><p class="c12-14"><span class="c12-2">Gel run after MiniPrep to check if plasmids are clean </span></p><p class="c12-9"><span>-</span><span> </span><span>1 %-agarose-1xTAE gel from yesterday (prepared by Jan)</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 3 μl 2 log ladder</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 1 μl plasmid + 1 μl 5xLD + 3 μl dH</span><span class="c12-10">2</span><span>O</span></p><p class="c12-9"><span>-</span><span> </span><span>run at 100 V</span></p><p class="c12-9"><span>-</span><span> </span><span>EtBr staining + destaining in water</span></p><p class="c12-9"><span>-</span><span> </span><span>UV detection </span></p><p class="c12-14 c12-43"><span>loading: Marker/Term</span><span class="c12-10">rev</span><span> C1/DarR</span><span class="c12-10">rev</span><span> C4/ RBS</span><span class="c12-10">rev</span><span> C1/part6.3 A C1/part6.3 B C1/ part6.3 B C2/ part6.3 B C3</span></p><img src="https://static.igem.org/mediawiki/2013/f/fb/Goe-21.08.13-RT-2.png" /><p class="c12-8"><span></span></p><p class="c12-15 c12-14"><span>→ all plasmids are purified from chrom.DNA and look normal</span></p><p class="c12-15 c12-14"><span>→ samples stored in DarR reporter system box</span><span class="c12-2">Re-streak of part 1 – 4 Clones 1 – 3 on LB</span><span class="c12-2 c12-5">Amp</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>plates from 6.6.13 (stored in fridge…. at 4 °C….)</span></p><p class="c12-9"><span>-</span><span> </span><span>bacteria were quite dry, so I scratched some cell material off and streaked it out on new plates</span></p><p class="c12-9"><span>-</span><span> </span><span>incubation ON at 37 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>let’s hope they grow…</span></p><p class="c12-9 c12-37"><span></span></p><p class="c12-14"><span class="c12-2">Preparation of new LB</span><span class="c12-2 c12-5">Cm</span><span class="c12-2"> plates</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>2x500 ml</span></p><p class="c12-9"><span>-</span><span> </span><span>35 μg/ml Cm</span></p><p class="c12-9"><span>-</span><span> </span><span>stored at 4 °C, big fridge</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">observation of clones of Ribo A C4 and C5 CFP under UV light</span></p><p class="c12-14"><span>(all clones with YFP don’t glow under UV light)</span><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-62">Pictures:</span></p> | ||
+ | <div style="display:inline-block"> | ||
+ | <p class="c12-14"><span>Ribo A C4 + CFP UV light</span></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/3/3c/Goe-21.08.13-RT-3.png" /> | ||
+ | <p class="c12-8"><span></span></p> | ||
+ | </div> | ||
+ | <div style="display:inline-block"> | ||
+ | <p class="c12-14"><span>Ribo A C4 + CFP transmitted light</span></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/e/e1/Goe-21.08.13-RT-4.png" /> | ||
+ | <p class="c12-8"><span></span></p> | ||
+ | </div> | ||
+ | <div style="display:inline-block"> | ||
+ | <p class="c12-14"><span>Ribo A C5 + CFP UV light</span></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/f/f3/Goe-21.08.13-RT-5.png" /> | ||
+ | <p class="c12-8"><span></span></p> | ||
+ | </div> | ||
+ | <div style="display:inline-block"> | ||
+ | <p class="c12-14"><span>Ribo A C5 + CFP transmiited light</span></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/8/82/Goe-21.08.13-RT-6.png" /> | ||
+ | <p class="c12-8"><span></span></p> | ||
+ | </div> | ||
+ | <div style="display:inline-block"> | ||
+ | <p class="c12-14"><span>Control w/o insert UV light</span></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/0/00/Goe-21.08.13-RT-7.png" /> | ||
+ | <p class="c12-8"><span></span></p> | ||
+ | </div> | ||
+ | <div style="display:inline-block"> | ||
+ | <p class="c12-14"><span>Control w/o insert transmitted light</span></p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/9/90/Goe-21.08.13-RT-8.png" /> | ||
+ | <p class="c12-8"><span></span></p> | ||
+ | </div> | ||
+ | <p class="c12-14"><span class="c12-2">Preparation of a Masterplate of 10 clones from plate with C4 and C5 CFP </span></p><p class="c12-14"><span class="c12-2">Inocculation of 10 clones from plate with C4 and C5 CFP in LB medium with Cm </span></p><p class="c12-14"><span class="c12-2">Colony PCR of the inoculated clones and a control w/o insert CFP</span></p><a href="#" name="5a777d4db43b88628de0e0305acf5ad15bd57b43"></a><a href="#" name="13"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-47"><p class="c12-22 c12-16 c12-14"><span class="c12-2">Component</span></p></td><td class="c12-23"><p class="c12-22 c12-16 c12-14"><span class="c12-2">Volume</span></p></td></tr><tr><td class="c12-47"><p class="c12-22 c12-16 c12-14"><span class="c12-2">Taq</span></p></td><td class="c12-23"><p class="c12-7 c12-22 c12-16"><span>1μl</span></p></td></tr><tr><td class="c12-47"><p class="c12-22 c12-16 c12-14"><span class="c12-2">10x Taq buffer</span></p></td><td class="c12-23"><p class="c12-7 c12-22 c12-16"><span>2,8μl</span></p></td></tr><tr><td class="c12-47"><p class="c12-22 c12-16 c12-14"><span class="c12-2">dNTP mix</span></p></td><td class="c12-23"><p class="c12-7 c12-22 c12-16"><span>1μl</span></p></td></tr><tr><td class="c12-47"><p class="c12-22 c12-16 c12-14"><span class="c12-2">iGEM_38 (VF2) 1:20</span></p></td><td class="c12-23"><p class="c12-7 c12-22 c12-16"><span>1μl</span></p></td></tr><tr><td class="c12-47"><p class="c12-22 c12-16 c12-14"><span class="c12-2">iGEM_39 (VR) 1:20</span></p></td><td class="c12-23"><p class="c12-7 c12-22 c12-16"><span>1μl</span></p></td></tr><tr><td class="c12-47"><p class="c12-22 c12-16 c12-14"><span class="c12-2">dH</span><span class="c12-1">2</span><span class="c12-2">O</span></p></td><td class="c12-23"><p class="c12-7 c12-22 c12-16"><span>18,8μl</span></p></td></tr><tr><td class="c12-47"><p class="c12-16 c12-14 c12-22"><span class="c12-2">Total</span></p></td><td class="c12-23"><p class="c12-7 c12-22 c12-16"><span>25μl</span></p></td></tr></tbody></table><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-8"><span class="c12-2"></span></p> | ||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | <p class="timeline-title goe-array">DNAse I digestion</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family: | ||
+ | "Arial","sans-serif";text-decoration:none">DNAse I digestion</span></h2> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">this | ||
+ | digestion is necessary to get rid of DNA contamination</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">use | ||
+ | PCR tubes</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">25 | ||
+ | </span><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">µl | ||
+ | sample</span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">2.5 </span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">µg RNA</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">2.5 | ||
+ | µl 10 x buffer</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">5 µl | ||
+ | DNAse I</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">x µl | ||
+ | RNAse free water</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">30 | ||
+ | min 37°C in the PCR cycler</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Stop | ||
+ | the reaction with 2.5 </span><span lang="EN-GB" style="font-size:11.0pt; | ||
+ | font-family:"Arial","sans-serif"">µl EDTA for 10 min at 65°C (also in the | ||
+ | cycler)</span></p> | ||
+ | <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family: | ||
+ | "Arial","sans-serif";text-decoration:none">Control PCR on digestion</span></h2> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">PCR | ||
+ | Scheme (50 </span><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">µl)</span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">10 </span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">µl HF buffer</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">2 µl | ||
+ | dNTPs Mix</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">4 µl | ||
+ | Primer Mix (here rpsE)</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">1 µl | ||
+ | RNA</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">0,5 | ||
+ | µl PhuS</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">32 µl | ||
+ | RNAse free H<sub>2</sub>0.</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">PCR | ||
+ | Programm</span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">98°C | ||
+ | 2 min</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">98°C | ||
+ | 35 sec</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">52°C | ||
+ | 35 sec</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">72°C | ||
+ | 45 sec</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">72°C | ||
+ | 10 min</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">15°C | ||
+ | hold</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif""> </span></p> | ||
+ | <p class="MsoListParagraph" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">run a | ||
+ | gel to check if everything went correctly</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">In | ||
+ | this case we did this digestion with the wt, GP1344 and GP1346. Additionally | ||
+ | we used a NTC and cDNA control.</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""><img width="288" height="312" id="Bild 5" src="https://static.igem.org/mediawiki/2013/5/54/Goe-arr-Image006.jpg" alt="Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130820_DNAse I digestion control PCR:Folie1.jpg"></span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Wingdings">Ø<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">The | ||
+ | digestion worked fine. No DNA detectable in our samples, only in the cDNA</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Wingdings">Ø<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">Determine | ||
+ | RNA concentration</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Wingdings">Ø<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><b><span lang="EN-GB" style="font-family:"Arial","sans-serif"">This | ||
+ | failed</span></b></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">We | ||
+ | did the digestion again. This time we used 6 samples in total, two samples of | ||
+ | each strain. IN the first three samples we used </span><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">5 µl DNAse I | ||
+ | according to protocol, in the other three samples we only used 2.5 µl DNAse | ||
+ | I.</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Stored | ||
+ | the samples over night at -20°C.</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""> </span></p> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
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+ | <div class="tlob" id="tl_0820"> | ||
+ | <span class="date">20th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <p class="timeline-title goe-rt">Gel extraction of DarR<sub>rev</sub> insert , Restriction digest of Term<sub>rev</sub> with PstI FD, Restriction digest of RBS<sub>rev</sub> C1 and part6.3 A and B (C2 each) with EcoRI FD, AP treatment of Term<sub>rev</sub> vector for ligation with DarR<sub>rev</sub> insert , Restriction Digest of all vectors digested with EcoRI FD today, ligation.....</p> | ||
+ | <div class="timeline-cont"> | ||
+ | |||
+ | <p class="c12-14"><span class="c12-2">Transformation of yesterday´s ligation</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Gel extraction of DarR</span><span class="c12-1">rev</span><span class="c12-2"> insert</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>with Qiagen PCR gel/PCR purification kit</span></p><p class="c12-9"><span>-</span><span> </span><span>since DarR</span><span class="c12-10">rev</span><span> insert has > 600 bp, no isopropanol was added/only QG buffer used</span></p><p class="c12-9"><span>-</span><span> </span><span>only 1 column used since <400 mg gel</span></p><p class="c12-9"><span>-</span><span> </span><span>elution 1x with 30 μl of pre-heated HPLC water, incubation at 50 °C for 2 min</span></p><p class="c12-9"><span>-</span><span> </span><span>NanoDrop concentration measurement </span></p><a href="#" name="2bee1d20bbac05195e25b0de69f38f9909dcf2b9"></a><a href="#" name="5"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-35"><p class="c12-7"><span class="c12-2">sample</span></p></td><td class="c12-33"><p class="c12-7"><span class="c12-2">ng/μl</span></p></td><td class="c12-58"><p class="c12-7"><span class="c12-2">A</span><span class="c12-1">260nm</span><span class="c12-2">/A</span><span class="c12-1">280nm</span></p></td><td class="c12-59"><p class="c12-7"><span class="c12-2">A</span><span class="c12-1">260nm</span><span class="c12-2">/A</span><span class="c12-1">230nm</span></p></td></tr><tr><td class="c12-35"><p class="c12-7"><span>DarR</span><span class="c12-10">rev</span><span> insert X+P</span></p></td><td class="c12-33"><p class="c12-7"><span>14.8</span></p></td><td class="c12-58"><p class="c12-7"><span>1.53</span></p></td><td class="c12-59"><p class="c12-7"><span>0.12</span></p></td></tr></tbody></table><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Restriction digest of Term</span><span class="c12-1">rev</span><span class="c12-2"> with PstI FD </span></p><p class="c12-9"><span>-</span><span> </span><span>DNA: Term</span><span class="c12-10">rev</span><span> C1 plasmid restricted with SpeI and purified yesterday</span></p><p class="c12-9"><span>-</span><span> </span><span>digest:</span><span class="c12-2"> </span></p><a href="#" name="f70c584bb0a8e6b3079aea2c9f470e679b2d5614"></a><a href="#" name="6"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">Component</span></p></td><td class="c12-44"><p class="c12-14"><span class="c12-2">Volume</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">plasmid</span></p></td><td class="c12-44"><p class="c12-7"><span>30μl (volume determined with pipet)</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">10x FD Green buffer</span></p></td><td class="c12-44"><p class="c12-7"><span>4μl</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">PstI FD</span></p></td><td class="c12-44"><p class="c12-7"><span>4μl</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">dH</span><span class="c12-1">2</span><span class="c12-2">O</span></p></td><td class="c12-44"><p class="c12-7"><span>2μl</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">Total</span></p></td><td class="c12-44"><p class="c12-7"><span>40μl</span></p></td></tr></tbody></table><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-9"><span>-</span><span> </span><span>digest for 2 h at 37 °C</span></p><p class="c12-9 c12-37"><span></span></p><p class="c12-14"><span class="c12-2">Restriction digest of RBS</span><span class="c12-1">rev</span><span class="c12-2"> C1 and part6.3 A and B (C2 each) with EcoRI FD </span></p><p class="c12-9"><span>-</span><span> </span><span>DNA: plasmids purified yesterday</span></p><p class="c12-9"><span>-</span><span> </span><span>digest for all reactions (pipette individually, no mastermix): </span></p><a href="#" name="5e4c9787eae85c6b28f95f69339c34d4c39b410e"></a><a href="#" name="7"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">Component</span></p></td><td class="c12-44"><p class="c12-14"><span class="c12-2">Volume</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">plasmid</span></p></td><td class="c12-44"><p class="c12-7"><span>8μl (since each plasmid had > 200 ng/μl)</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">10x FD Green buffer</span></p></td><td class="c12-44"><p class="c12-7"><span>4μl</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">Eco</span></p><p class="c12-14"><span class="c12-2">I FD</span></p></td><td class="c12-44"><p class="c12-7"><span>4μl</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">dH</span><span class="c12-1">2</span><span class="c12-2">O</span></p></td><td class="c12-44"><p class="c12-7"><span>24μl</span></p></td></tr><tr><td class="c12-11"><p class="c12-14"><span class="c12-2">Total</span></p></td><td class="c12-44"><p class="c12-7"><span>40μ</span></p></td></tr></tbody></table><p class="c12-9"><span> -</span><span> </span><span>digest for 2 h at 37 °C</span></p><p class="c12-9 c12-37"><span></span></p><p class="c12-14"><span class="c12-2">Sequencing</span></p><p class="c12-14"><span class="c12-2"> results RBS</span><span class="c12-1">rev</span><span class="c12-2"> C1 and part6.3 A and B (C1 and C2 each) </span></p><p class="c12-9"><span>-</span><span> </span><span class="c12-2">part 6.3 A C1 (sample 3) </span><span>– sequence as expected (no mutations)</span></p><p class="c12-9"><span>-</span><span> </span><span class="c12-2">part 6.3 A C2 (sample 4) </span><span>– a G at position 52 of Hybridization oligo is missing → digest sample (see above) thrown away, new digest (exactly as described above) using clone 1</span></p><p class="c12-9"><span>-</span><span> </span><span class="c12-2">part 6.3 B C1 (sample 5)</span><span> – sequencing did not work, only first part of oligo in vector sequenced → was ok</span></p><p class="c12-9"><span>-</span><span> </span><span class="c12-2">part 6.3 B C2 (sample 6)</span><span> – sequencing did not work, only first part of oligo in vector sequenced → was ok → further digest</span></p><p class="c12-9"><span>-</span><span> </span><span class="c12-2">RBS</span><span class="c12-1">rev</span><span class="c12-2"> in pSB1C3 C1 (sample 7) </span><span>– sequence as expected</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Loading:</span><span> </span></p><p class="c12-14"><span>Marker/uncut Term</span><span class="c12-10">rev</span><span> C1/RD Term</span><span class="c12-10">rev</span><span> C1/uncut RBS</span><span class="c12-10">rev</span><span> C1/RD RBS</span><span class="c12-10">rev</span><span> C1/uncut part6.3 A C1/RD part6.3 A C1/uncut part6.3 B C2/RD part6.3 B C2/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/b/b3/Goe-20.08.13-RT-1.png" /><p class="c12-9"><span>-</span><span> </span><span>all digests are only partial, but most of plasmid is cut → purify digests</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">New sizes of Riboswitch PCR products</span></p><p class="c12-14"><span> </span></p><p class="c12-14"><span>I checked again the alignments of the riboswitch PCR products, and the sizes I entered into the journal on 24.6.13 are partially wrong. </span></p><p class="c12-14"><span> </span></p><p class="c12-14"><span>The correct sizes are:</span><span class="c12-2"> </span></p><a href="#" name="192b80c14d9fabf51c53dcc15dc94e0a893fb6be"></a><a href="#" name="8"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-53"><p class="c12-14"><span class="c12-2"> </span></p></td><td class="c12-36"><p class="c12-14"><span class="c12-2">Primers</span></p></td><td class="c12-45"><p class="c12-14"><span class="c12-2">approx. PCR product size (w/o prefix/suffix)</span></p></td><td class="c12-49"><p class="c12-14"><span class="c12-2">approx. PCR product size (w/ prefix/suffix)</span></p></td></tr><tr><td class="c12-53"><p class="c12-14"><span class="c12-2">Riboswitch with native Promoter and RBS</span></p></td><td class="c12-36"><p class="c12-7"><span>iGEM_42</span></p><p class="c12-7"><span>iGEM_43</span></p></td><td class="c12-45"><p class="c12-7"><span>460 bp</span></p></td><td class="c12-49"><p class="c12-7"><span>500 bp</span></p></td></tr><tr><td class="c12-53"><p class="c12-14"><span class="c12-2">Riboswitch with native Promoter</span></p></td><td class="c12-36"><p class="c12-7"><span>iGEM_42</span></p><p class="c12-7"><span>iGEM_41</span></p></td><td class="c12-45"><p class="c12-7"><span>315 bp</span></p></td><td class="c12-49"><p class="c12-7"><span>355 bp</span></p></td></tr><tr><td class="c12-53"><p class="c12-14"><span class="c12-2">Riboswitch only</span></p></td><td class="c12-36"><p class="c12-7"><span>iGEM_40</span></p><p class="c12-7"><span>iGEM_41</span></p></td><td class="c12-45"><p class="c12-7"><span>230 bp</span></p></td><td class="c12-49"><p class="c12-7"><span>270 bp</span></p></td></tr><tr><td class="c12-53"><p class="c12-14"><span class="c12-2">Riboswitch with native RBS</span></p></td><td class="c12-36"><p class="c12-7"><span>iGEM_40</span></p><p class="c12-7"><span>iGEM_43</span></p></td><td class="c12-45"><p class="c12-7"><span>375 bp</span></p></td><td class="c12-49"><p class="c12-7"><span>415 bp</span></p></td></tr></tbody></table><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2">Inoculation of clones for CryoStocks </span></p><p class="c12-14"><span>inoculation of</span></p><p class="c12-9"><span>-</span><span> </span><span>RBS</span><span class="c12-10">rev</span><span> + pSB1C3 C1</span></p><p class="c12-9"><span>-</span><span> </span><span>part 6.3 A C1</span></p><p class="c12-9"><span>-</span><span> </span><span>part 6.3 B C1, C2, C3</span></p><p class="c12-9"><span>-</span><span> </span><span>Term</span><span class="c12-10">rev</span><span> + pSB1C3 C1</span></p><p class="c12-9"><span>-</span><span> </span><span>DarR</span><span class="c12-10">rev</span><span> + pSB1C3 C4</span></p><p class="c12-14 c12-43"><span> → from master plates, inoculation in 4 ml LB</span><span class="c12-5">Cm</span></p><p class="c12-14"><span>incubation ON at 37 °C, 200 – 210 rpm</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Purification of vectors digested today</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>samples: RBS</span><span class="c12-10">rev</span><span> + pSB1C3 RD, part 6.3 A C1 and part 6.3 B C2 RD, Term</span><span class="c12-10">rev</span><span> + pSB1C3 RD</span></p><p class="c12-9"><span>-</span><span> </span><span>with Qiagen PCR clean-up kit</span></p><p class="c12-9"><span>-</span><span> </span><span>addition of 500 μl PB buffer to each reaction</span></p><p class="c12-9"><span>-</span><span> </span><span>elution with 30 μl pre-warmend HPLC water, incubation at 50 °C for 2 min</span></p><p class="c12-8"><span></span></p><p class="c12-14 c12-24"><span class="c12-2">AP treatment of Term</span><span class="c12-1">rev</span><span class="c12-2"> vector for ligation with DarR</span><span class="c12-1">rev</span><span class="c12-2"> insert</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>this is silly… I could have done it before purification, then I would have lost less vector…</span></p><p class="c12-9"><span>-</span><span> </span><span>addition of 1 μl AP, 4 μl AP buffer 10x, 8 μl dH</span><span class="c12-10">2</span><span>O to 27 μl purified vector</span></p><p class="c12-9"><span>-</span><span> </span><span>incubation for 1 h at 37 °C (PstI makes 3’ overhang)</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Restriction Digest of all vectors digested with EcoRI FD today </span></p><p class="c12-9"><span>-</span><span> </span><span>samples: RBS</span><span class="c12-10">rev</span><span> + pSB1C3 RD, part 6.3 A C1 and part 6.3 B C2 RD</span></p><p class="c12-9"><span>-</span><span> </span><span>all samples had a volume of 30 μl after clean-up, so addition of 2 μl dH</span><span class="c12-10">2</span><span>O and 4 μl 10x FD buffer</span></p><p class="c12-9"><span>-</span><span> </span><span>addition of 4 μl SpeI FD to RBS</span><span class="c12-10">rev</span><span> + pSB1C3 RD sample, and 4μl XbaI FD to part 6.3 A C1 and part 6.3 B C2 RD samples</span></p><p class="c12-9"><span>-</span><span> </span><span>for all reactions: total volume of 40 μl</span></p><p class="c12-9"><span>-</span><span> </span><span>part 6.3 A C1 and part 6.3 B C2 RD digests incubated at 37 °C for 1 h (then dephosphorylation)</span></p><p class="c12-9"><span>-</span><span> </span><span>RBS</span><span class="c12-10">rev</span><span> + pSB1C3 RD digest incubated at 37 °C for 2 h (no dephosphorylation, since Promoter hybridization oligos are dephosphorylated!)</span></p><p class="c12-14"><span class="c12-2">AP treatment of part 6.3 A C1 and B C2 </span></p><p class="c12-9"><span>-</span><span> </span><span>40 μl reaction + 5 μl 10x AP buffer + 2 μl AP + 3 μl dH</span><span class="c12-10">2</span><span>O</span></p><p class="c12-9"><span>-</span><span> </span><span>incubation for ca. 50 min at 37 °C (although only 15 min needed, since XbaI and EcoRI generate 5’ overhangs. But dephosphorylation often incomplete…)</span></p><p class="c12-14"><span class="c12-2">Gel run of digested and AP treated part 6.3 A C1 and BC2 and RBS</span><span class="c12-1">rev</span><span class="c12-2"> digested with SpeI and EcoRI </span></p><p class="c12-9"><span>-</span><span> </span><span>1%-agarose-1xTAE gel</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 3 μl 2 log ladder</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 4 μl of dephosphorylated reactions + 1 μl 5xLD</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 3 μl RBS</span><span class="c12-10">rev</span><span> digest + 1 μl dH</span><span class="c12-10">2</span><span>O + 1μl 5xLD</span></p><p class="c12-9"><span>-</span><span> </span><span>run at 100 V</span></p><p class="c12-9"><span>-</span><span> </span><span>EtBr staining + destaining in water</span></p><p class="c12-9"><span>-</span><span> </span><span>UV detection</span></p><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2">Loading: </span></p><p class="c12-14"><span class="c12-2">Marker/</span><span> uncut RBS</span><span class="c12-10">rev</span><span> C1/RD RBS</span><span class="c12-10">rev</span><span> C1/uncut part6.3 A C1/RD + AP part6.3 A C1/uncut part6.3 B C2/RD + AP part6.3 B C2/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/7/79/Goe-20.08.13-RT-2.png" /><p class="c12-9"><span>-</span><span> </span><span>no over-digest; digests still incomplete, but since AP treatment of at least part 6.3 A and B vector, this should not matter; in case of RBS</span><span class="c12-10">rev</span><span> ligation, re-ligands could occur</span></p><p class="c12-9 c12-37"><span></span></p><p class="c12-14"><span class="c12-2">Purification of digested and dephosphorylated samples </span></p><p class="c12-9"><span>-</span><span> </span><span>with Qiagen PCR purification kit</span></p><p class="c12-9"><span>-</span><span> </span><span>addition of 500 μl PB buffer to each sample</span></p><p class="c12-9"><span>-</span><span> </span><span>elution 1x with 30 μl HPLC water (pre-warmed), incubation for 2 min at 50 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>NanoDrop concentration measurement</span></p><img src="https://static.igem.org/mediawiki/2013/d/d4/Goe-20.08.13-RT-3.png" /><p class="c12-14"><span> </span></p><p class="c12-14"><span class="c12-2">Ligation</span></p><p class="c12-14"><span class="c12-2">Ligation of</span></p><p class="c12-14"><span class="c12-2">a) DarR</span><span class="c12-1">rev</span><span class="c12-2"> insert + Term</span><span class="c12-1">rev</span><span class="c12-2"> vector → Term</span><span class="c12-1">rev</span><span class="c12-2">-DarR</span><span class="c12-1">rev</span><span class="c12-2"> in pSB1C3</span></p><p class="c12-14"><span class="c12-2">b) RBS</span><span class="c12-1">rev</span><span class="c12-2"> hybridization oligo + part6.3 A/part 6.3 B → part 6.4 A/part 6.4 B (RBS</span><span class="c12-1">rev</span><span class="c12-2">-Promoter1/3</span><span class="c12-1">rev</span><span class="c12-2"> in part 6.2)</span></p><p class="c12-14"><span class="c12-2">c) Promoter1/3</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 derived from RBS</span><span class="c12-1">rev</span><span class="c12-2"> in pSB1C3 digest → Promoter1/3</span><span class="c12-1">rev</span><span class="c12-2"> in pSB1C3</span></p><p class="c12-14"><span> </span></p><p class="c12-14"><span class="c12-62">Pipetting scheme</span></p><p class="c12-14"><span>a) DarR</span><span class="c12-10">rev</span><span> insert + Term</span><span class="c12-10">rev</span><span> vector → Term</span><span class="c12-10">rev</span><span>-DarR</span><span class="c12-10">rev</span><span> in pSB1C3</span></p><p class="c12-14"><span>Uni D’dorf ligation calculator: for a ratio vector:insert = 1:3, one has to use 50 ng of a 2200 bp (= 2070 of pSB1C3 + 130 bp of terminator) vector and 44 ng insert of 650 bp → approximate amounts used in this ligation.</span></p><a href="#" name="0fb509d8e043af48238ba8e06f590cf8a231ea0f"></a><a href="#" name="9"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-31"><p class="c12-14"><span class="c12-2">Component</span></p></td><td class="c12-30"><p class="c12-7"><span class="c12-2">With DarR</span><span class="c12-1">rev</span><span class="c12-2"> insert</span></p></td><td class="c12-26"><p class="c12-7"><span class="c12-2">Without insert control</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>Term</span><span class="c12-10">rev</span><span> vector (purified today, 19.9 ng/µl)</span></p></td><td class="c12-30"><p class="c12-7"><span>3 µl</span></p></td><td class="c12-26"><p class="c12-7"><span>3 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>DarR</span><span class="c12-10">rev</span><span> insert (purified today, 14.8 ng/µl) or dH</span><span class="c12-10">2</span><span>O for w/o insert control</span></p></td><td class="c12-30"><p class="c12-7"><span>3 µl</span></p></td><td class="c12-26"><p class="c12-7"><span>3 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>T4 ligase (ThermoScientific)</span></p></td><td class="c12-30"><p class="c12-7"><span>1 µl</span></p></td><td class="c12-26"><p class="c12-7"><span>1 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>10x T4 ligase buffer (ThermoScientific)</span></p></td><td class="c12-30"><p class="c12-7"><span>1 µl</span></p></td><td class="c12-26"><p class="c12-7"><span>1 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>dH</span><span class="c12-10">2</span><span>O</span></p></td><td class="c12-30"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-26"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>Total</span></p></td><td class="c12-30"><p class="c12-7"><span>10 µl</span></p></td><td class="c12-26"><p class="c12-7"><span>10 µl</span></p></td></tr></tbody></table><p class="c12-14"><span> </span></p><p class="c12-14"><span>b) RBS</span><span class="c12-10">rev</span><span> hybridization oligo + part6.3 A/part 6.3 B → part 6.4 A/part 6.4 B (RBS</span><span class="c12-10">rev</span><span>-Promoter1/3</span><span class="c12-10">rev</span><span> in part 6.2)</span></p><a href="#" name="e5c334fc1ffc797e801ec5511e0f87b49901ee7e"></a><a href="#" name="10"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-31"><p class="c12-14"><span class="c12-2">Component</span></p></td><td class="c12-26"><p class="c12-7"><span class="c12-2">With RBS</span><span class="c12-1">rev</span><span class="c12-2"> insert</span></p></td><td class="c12-30"><p class="c12-7"><span class="c12-2">Without insert control</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>Part 6.3 A vector (purified today, 30.5 ng/µl)</span></p></td><td class="c12-26"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>RBS</span><span class="c12-10">rev</span><span> hybridization oligo (prepared on 19.8.13) or dH</span><span class="c12-10">2</span><span>O for w/o insert control</span></p></td><td class="c12-26"><p class="c12-7"><span>8 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>8 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>T4 ligase (ThermoScientific)</span></p></td><td class="c12-26"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>10x T4 ligase buffer (ThermoScientific)</span></p></td><td class="c12-26"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>dH</span><span class="c12-10">2</span><span>O</span></p></td><td class="c12-26"><p class="c12-7"><span>6 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>6 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>Total</span></p></td><td class="c12-26"><p class="c12-7"><span>20 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>20 µl</span></p></td></tr></tbody></table><p class="c12-14"><span> </span></p><a href="#" name="cfedce22386b71456328ddc04adb3d94202922dc"></a><a href="#" name="11"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-31"><p class="c12-14"><span class="c12-2">Component</span></p></td><td class="c12-26"><p class="c12-7"><span class="c12-2">With RBS</span><span class="c12-1">rev</span><span class="c12-2"> insert</span></p></td><td class="c12-30"><p class="c12-7"><span class="c12-2">Without insert control</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>Part 6.3 B vector (purified today, 33.6 ng/µl)</span></p></td><td class="c12-26"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>RBS</span><span class="c12-10">rev</span><span> hybridization oligo (prepared on 19.8.13) or dH</span><span class="c12-10">2</span><span>O for w/o insert control</span></p></td><td class="c12-26"><p class="c12-7"><span>8 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>8 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>T4 ligase (ThermoScientific)</span></p></td><td class="c12-26"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>10x T4 ligase buffer (ThermoScientific)</span></p></td><td class="c12-26"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>dH</span><span class="c12-10">2</span><span>O</span></p></td><td class="c12-26"><p class="c12-7"><span>6 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>6 µl</span></p></td></tr><tr><td class="c12-31"><p class="c12-14"><span>Total</span></p></td><td class="c12-26"><p class="c12-7"><span>20 µl</span></p></td><td class="c12-30"><p class="c12-7"><span>20 µl</span></p></td></tr></tbody></table><p class="c12-14"><span> </span></p><p class="c12-14"><span>c) Promoter1/3</span><span class="c12-10">rev</span><span> + pSB1C3 derived from RBS</span><span class="c12-10">rev</span><span> in pSB1C3 digest → Promoter1/3</span><span class="c12-10">rev</span><span> in pSB1C3</span></p><a href="#" name="cb86a02a31ac20a6441ff413dc042c4ddc18cba1"></a><a href="#" name="12"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-13"><p class="c12-14"><span class="c12-2">Component</span></p></td><td class="c12-32"><p class="c12-7"><span class="c12-2">With Promoter1</span><span class="c12-1">rev</span><span class="c12-2"> insert</span></p></td><td class="c12-39"><p class="c12-7"><span class="c12-2">With Promoter3</span><span class="c12-1">rev</span><span class="c12-2"> insert</span></p></td><td class="c12-11"><p class="c12-7"><span class="c12-2">Without insert control</span></p></td></tr><tr><td class="c12-13"><p class="c12-14"><span>psB1C3 vector (derived from RBS</span><span class="c12-10">rev</span><span> C1 plasmid, purified today, 29.9 ng/µl)</span></p></td><td class="c12-32"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-39"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-11"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-13"><p class="c12-14"><span>Promoter1/3</span><span class="c12-10">rev</span><span> hybridization oligo (prepared on 19.8.13) or dH</span><span class="c12-10">2</span><span>O for w/o insert control</span></p></td><td class="c12-32"><p class="c12-7"><span>10 µl</span></p></td><td class="c12-39"><p class="c12-7"><span>10 µl</span></p></td><td class="c12-11"><p class="c12-7"><span>10 µl</span></p></td></tr><tr><td class="c12-13"><p class="c12-14"><span>T4 ligase (ThermoScientific)</span></p></td><td class="c12-32"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-39"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-11"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-13"><p class="c12-14"><span>10x T4 ligase buffer (ThermoScientific)</span></p></td><td class="c12-32"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-39"><p class="c12-7"><span>2 µl</span></p></td><td class="c12-11"><p class="c12-7"><span>2 µl</span></p></td></tr><tr><td class="c12-13"><p class="c12-14"><span>dH</span><span class="c12-10">2</span><span>O</span></p></td><td class="c12-32"><p class="c12-7"><span>4 µl</span></p></td><td class="c12-39"><p class="c12-7"><span>4 µl</span></p></td><td class="c12-11"><p class="c12-7"><span>4 µl</span></p></td></tr><tr><td class="c12-13"><p class="c12-14"><span>Total</span></p></td><td class="c12-32"><p class="c12-7"><span>20 µl</span></p></td><td class="c12-39"><p class="c12-7"><span>20 µl</span></p></td><td class="c12-11"><p class="c12-7"><span>20 µl</span></p></td></tr></tbody></table><p class="c12-14"><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>All reaction were pipetted individually/no mastermix</span></p><p class="c12-9"><span>-</span><span> </span><span>Incubation ON at 16 °C (cold room heat block)</span></p><p class="c12-8"><span></span></p> | ||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | <p class="timeline-title goe-array">DNAse I digestion</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family: | ||
+ | "Arial","sans-serif";text-decoration:none">DNAse I digestion</span></h2> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">this | ||
+ | digestion is necessary to get rid of DNA contamination</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">use | ||
+ | PCR tubes</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">25 | ||
+ | </span><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">µl | ||
+ | sample</span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">2.5 </span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">µg RNA</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">2.5 | ||
+ | µl 10 x buffer</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">5 µl | ||
+ | DNAse I</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">x µl | ||
+ | RNAse free water</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">30 | ||
+ | min 37°C in the PCR cycler</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Stop | ||
+ | the reaction with 2.5 </span><span lang="EN-GB" style="font-size:11.0pt; | ||
+ | font-family:"Arial","sans-serif"">µl EDTA for 10 min at 65°C (also in the | ||
+ | cycler)</span></p> | ||
+ | <h2><span lang="EN-GB" style="font-size:11.0pt;line-height:115%;font-family: | ||
+ | "Arial","sans-serif";text-decoration:none">Control PCR on digestion</span></h2> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">PCR | ||
+ | Scheme (50 </span><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">µl)</span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">10 </span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">µl HF buffer</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">2 µl | ||
+ | dNTPs Mix</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">4 µl | ||
+ | Primer Mix (here rpsE)</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">1 µl | ||
+ | RNA</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">0,5 | ||
+ | µl PhuS</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">32 µl | ||
+ | RNAse free H<sub>2</sub>0.</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">PCR | ||
+ | Programm</span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">98°C | ||
+ | 2 min</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">98°C | ||
+ | 35 sec</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">52°C | ||
+ | 35 sec</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">72°C | ||
+ | 45 sec</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">72°C | ||
+ | 10 min</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">15°C | ||
+ | hold</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif""> </span></p> | ||
+ | <p class="MsoListParagraph" style=""><span lang="EN-GB" style="font-family:Symbol">·<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">run a | ||
+ | gel to check if everything went correctly</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">In | ||
+ | this case we did this digestion with the wt, GP1344 and GP1346. Additionally | ||
+ | we used a NTC and cDNA control.</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""><img width="288" height="312" id="Bild 5" src="https://static.igem.org/mediawiki/2013/5/54/Goe-arr-Image006.jpg" alt="Macintosh HD:Users:jangundlach:Dropbox:Sam, Jan:LabBook:Bilder LabbBook 1:130820_DNAse I digestion control PCR:Folie1.jpg"></span></p> | ||
+ | <p class="MsoListParagraphCxSpFirst" style=""><span lang="EN-GB" style="font-family:Wingdings">Ø<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">The | ||
+ | digestion worked fine. No DNA detectable in our samples, only in the cDNA</span></p> | ||
+ | <p class="MsoListParagraphCxSpMiddle" style=""><span lang="EN-GB" style="font-family:Wingdings">Ø<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-GB" style="font-family:"Arial","sans-serif"">Determine | ||
+ | RNA concentration</span></p> | ||
+ | <p class="MsoListParagraphCxSpLast" style=""><span lang="EN-GB" style="font-family:Wingdings">Ø<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><b><span lang="EN-GB" style="font-family:"Arial","sans-serif"">This | ||
+ | failed</span></b></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">We | ||
+ | did the digestion again. This time we used 6 samples in total, two samples of | ||
+ | each strain. IN the first three samples we used </span><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">5 µl DNAse I | ||
+ | according to protocol, in the other three samples we only used 2.5 µl DNAse | ||
+ | I.</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-GB" style="font-size:11.0pt;font-family:"Arial","sans-serif"">Stored | ||
+ | the samples over night at -20°C.</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Arial","sans-serif""> </span></p> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="tlob" id="tl_0819"> | ||
+ | <span class="date">19th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <p class="timeline-title goe-rt">Mini-Preps of RiboA Clones 1, 2, 4 and 5 (see gel from 12.08.), RD of Ribo 2,4,5, Ligations of the Riboswitches into next vectors, MiniPrep of RBS<sub>rev</sub> + pSB1C3, Prom1<sub>rev</sub> + part6.2, Prom3<sub>rev</sub> + part 6.2 (C1 – C3 each) and PCR test, PCR to test, if RBS<sub>rev</sub> C1 – C3 are positive or negative, Test restriction digest of Prom1<sub>rev</sub> + part6.2, Prom3<sub>rev</sub> + part 6.2 (C1 – C3 each)....</p> | ||
+ | <div class="timeline-cont"> | ||
+ | |||
+ | <p class="c12-14 c12-24"><span class="c12-2">Cloning of Riboswitch A </span></p><p class="c12-14 c12-24"><span class="c12-2">Mini-Preps of RiboA Clones 1, 2, 4 and 5 (see gel from 12.08.) using the plasmid purification kit</span></p><p class="c12-14 c12-24"><span>Nanodrop measurements:</span></p><a href="#" name="1125b986674db083e9c9e43e752e86645c46af29"></a><a href="#" name="0"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-54"><p class="c12-4"><span class="c12-2">Sample</span></p></td><td class="c12-52"><p class="c12-4"><span class="c12-2">ng/µl</span></p></td><td class="c12-38"><p class="c12-4"><span class="c12-2">A</span><span class="c12-1">260nm</span><span class="c12-2">/A</span><span class="c12-1">280nm</span></p></td><td class="c12-41"><p class="c12-4"><span class="c12-2">A</span><span class="c12-1">260nm</span><span class="c12-2">/A</span><span class="c12-1">230nm</span></p></td></tr><tr><td class="c12-54"><p class="c12-4"><span>Ribo A clone 1</span></p></td><td class="c12-52"><p class="c12-4"><span>213.9</span></p></td><td class="c12-38"><p class="c12-4"><span>1.89</span></p></td><td class="c12-41"><p class="c12-4"><span>1.86</span></p></td></tr><tr><td class="c12-54"><p class="c12-4"><span>Ribo A clone 2</span></p></td><td class="c12-52"><p class="c12-4"><span>152.5</span></p></td><td class="c12-38"><p class="c12-4"><span>1.95</span></p></td><td class="c12-41"><p class="c12-4"><span>2.00</span></p></td></tr><tr><td class="c12-54"><p class="c12-4"><span>Ribo A clone 4</span></p></td><td class="c12-52"><p class="c12-4"><span>396.6</span></p></td><td class="c12-38"><p class="c12-4"><span>1.90</span></p></td><td class="c12-41"><p class="c12-4"><span>2.11</span></p></td></tr><tr><td class="c12-54"><p class="c12-4"><span>Ribo A clone 5</span></p></td><td class="c12-52"><p class="c12-4"><span>429.4</span></p></td><td class="c12-38"><p class="c12-4"><span>1.89</span></p></td><td class="c12-41"><p class="c12-4"><span>2.18</span></p></td></tr></tbody></table><p class="c12-14 c12-24"><span class="c12-2"> </span></p><p class="c12-14 c12-24"><span class="c12-2">Restriction Digestion of</span></p><p class="c12-14 c12-24"><span class="c12-2">Ribo A clones 4 +5, </span><span>(R.D. with E + S)</span></p><p class="c12-14 c12-24"><span class="c12-2">Ribo B clone 2, </span><span>(R.D. with X + P)</span></p><p class="c12-14 c12-24"><span class="c12-2">Ribo C clone 4, </span><span>(R.D. with E + S)</span></p><p class="c12-14 c12-24"><span class="c12-2">Ribo D clone 5</span><span> (R.D. with X + P)</span></p><p class="c12-14 c12-24"><span class="c12-2"> </span></p><p class="c12-14 c12-24"><span>1500 ng DNA</span></p><p class="c12-14 c12-24"><span>3µl enzyme FD</span></p><p class="c12-14 c12-24"><span>3µl enzyme FD</span></p><p class="c12-14 c12-24"><span>4µl Buffer FD</span></p><p class="c12-14 c12-24"><span>40 µl reaction (filled up with H</span><span class="c12-10">2</span><span>O)</span></p><p class="c12-14 c12-24"><span>for 1.5 h at 37°C</span></p><p class="c12-14 c12-24"><span> </span></p><p class="c12-14 c12-24"><span class="c12-2">Gel:</span></p><p class="c12-14 c12-24"><span class="c12-2">M/plasmid DNA Ribo A clone 4/ R.D. Ribo A clone 4/ plasmid DNA Ribo A clone 5/ R.D. Ribo A clone 5/ plasmid DNA Ribo B clone 2/ R.D. Ribo B clone 2/ plasmid DNA Ribo C clone 4/ R.D. Ribo C clone 4/ plasmid DNA Ribo D clone 5/ R.D. Ribo D clone 5</span></p><img src="https://static.igem.org/mediawiki/2013/c/c12-2/Goe-19.08.13-RT-1.png" /><p class="c12-8 c12-24"><span class="c12-2"></span></p><p class="c12-14 c12-24"><span>→ loading of all R.D. Ribos A-D onto Gel for gel extraction </span></p><p class="c12-14 c12-24"><span>→ Gelextraxtion using the PCR purification kit, nanodrop measurements on the eppis (3-5ng/µl)</span></p><p class="c12-8 c12-24"><span></span></p><p class="c12-14 c12-24"><span class="c12-2">Ligations of the Riboswitches into next vectors </span></p><p class="c12-14 c12-24"><span>Ribo A clones 4 +5, (R.D. with E + S) with CFP and YFP (R.D. with E+X) (see 12.08.)</span></p><p class="c12-14 c12-24"><span>Ribo B clone 2, (R.D. with X + P) with Part1 and Part3 (R.D. with S+P) (see 12.08.)</span></p><p class="c12-14 c12-24"><span>Ribo C clone 4, (R.D. with E + S) with Part8 (R.D. with E+X) (see 12.08.)</span></p><p class="c12-14 c12-24"><span>Ribo D clone 5 (R.D. with X + P) with Part1 and Part3 (R.D. with S+P) (see 12.08.) </span></p><p class="c12-14 c12-24"><span>For that, all of the inserts (60ng) was used for ligation ino the Vectors (20ng) </span></p><p class="c12-14 c12-43"><span>0,75-1µl Vectors</span></p><p class="c12-14 c12-43"><span>12µl Insert (Ribo A-D)</span></p><p class="c12-14 c12-43"><span>2µl T4 Buffer</span></p><p class="c12-14 c12-43"><span>2µl T4 Ligase</span></p><p class="c12-14 c12-43"><span>3-3,25µl H20</span></p><p class="c12-14 c12-43"><span>20µl reaction </span></p><p class="c12-14 c12-24"><span>Incubation overnight at 16°C</span></p><p class="c12-8 c12-24"><span></span></p><p class="c12-14 c12-24"><span class="c12-2">MiniPrep of RBS</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3, Prom1</span><span class="c12-1">rev</span><span class="c12-2"> + part6.2, Prom3</span><span class="c12-1">rev</span><span class="c12-2"> + part 6.2 (C1 – C3 each) </span></p><p class="c12-9"><span>-</span><span> </span><span>kit: Nucleospin, Macherey-Nagel</span></p><p class="c12-9"><span>-</span><span> </span><span>1</span><span class="c12-5">st</span><span> elution: 30 μl HPLC-water (pre-warmed), incubation for 2 min at 50 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>2</span><span class="c12-5">nd</span><span> elution: 22 μl HPLC-water (pre-warmed), incubation for 2 min at 50 °C</span></p><p class="c12-9"><span>-</span><span> </span><span>no concentration measurement, since NanoDrop is dirty and measures 6 – 7 ng DNA in HPLC water used as a blank before…</span></p><p class="c12-9"><span>-</span><span> </span><span>samples stored in “DarR reporter system” - box</span></p><p class="c12-3"><span></span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">PCR to test, if RBS</span><span class="c12-1">rev</span><span class="c12-2"> C1 – C3 are positive or negative</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>RBS</span><span class="c12-10">rev</span><span> insert has only 12 bp → one would not see it in a gel</span></p><p class="c12-9"><span>-</span><span> </span><span>in Colony PCR, there was for all clones a band at height of terminator band (re-ligand)</span></p><p class="c12-9"><span>-</span><span> </span><span>perform PCR to test, if terminator band is really there/ if clones are positive</span></p><p class="c12-9"><span>-</span><span> </span><span>PCR (similar to colony PCR, but with plasmids as a template):</span></p><p class="c12-3"><span></span></p><a href="#" name="7fe2bd2d13d171a3c9284fc10b2cae22ea6ce0c3"></a><a href="#" name="1"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-51"><p class="c12-16 c12-14"><span class="c12-2">Component</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span class="c12-2">Volume</span></p></td></tr><tr><td class="c12-51"><p class="c12-16 c12-14"><span class="c12-2">Taq</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span>1 μl</span></p></td></tr><tr><td class="c12-51"><p class="c12-16 c12-14"><span class="c12-2">10x Taq buffer</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span>2.5 μl</span></p></td></tr><tr><td class="c12-51"><p class="c12-16 c12-14"><span class="c12-2">dNTP mix</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span>1 μl</span></p></td></tr><tr><td class="c12-51"><p class="c12-16 c12-14"><span class="c12-2">iGEM_38 (VF2) 1:20</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span>1 μl</span></p></td></tr><tr><td class="c12-51"><p class="c12-14 c12-16"><span class="c12-2">iGEM_39 (VR) 1:20</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span>1 μl</span></p></td></tr><tr><td class="c12-51"><p class="c12-16 c12-14"><span class="c12-2">dH</span><span class="c12-1">2</span><span class="c12-2">O</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span>17.5 μl</span></p></td></tr><tr><td class="c12-51"><p class="c12-16 c12-14"><span class="c12-2">template</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span>1 μl</span></p></td></tr><tr><td class="c12-51"><p class="c12-16 c12-14"><span class="c12-2">Total</span></p></td><td class="c12-12"><p class="c12-7 c12-16"><span>25 μl</span></p></td></tr></tbody></table><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-9"><span>-</span><span> </span><span>of the plasmids prepped today (see MiniPrep), a 1:20 dilution was prepared, of this dilution, 1 μl was used a template (1 reaction for each clone)</span></p><p class="c12-9"><span>-</span><span> </span><span>as an additional control, part 7 C1 plasmid (dilution from 15.6.13, 1:10) was used</span></p><p class="c12-9"><span>-</span><span> </span><span>Neg. control: 1 μl water as a template</span></p><p class="c12-9"><span>-</span><span> </span><span>preparation of a 6x MasterMix:</span></p><p class="c12-3"><span></span></p><p class="c12-14"><span> </span></p><a href="#" name="e36506b49dad64a763ef3905bc5a07c7901d0766"></a><a href="#" name="2"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-21"><p class="c12-18 c12-14"><span class="c12-2">Component</span></p></td><td class="c12-20"><p class="c12-18 c12-14"><span class="c12-2">Volum</span></p></td></tr><tr><td class="c12-21"><p class="c12-18 c12-14"><span class="c12-2">Taq</span></p></td><td class="c12-20"><p class="c12-0"><span>6μl</span></p></td></tr><tr><td class="c12-21"><p class="c12-18 c12-14"><span class="c12-2">10x Taq buffe</span></p></td><td class="c12-20"><p class="c12-0"><span>15μl</span></p></td></tr><tr><td class="c12-21"><p class="c12-18 c12-14"><span class="c12-2">dNTP mix</span></p></td><td class="c12-20"><p class="c12-0"><span>6μl</span></p></td></tr><tr><td class="c12-21"><p class="c12-18 c12-14"><span class="c12-2">iGEM_38 (V2) 1:20</span></p></td><td class="c12-20"><p class="c12-0"><span>6μl</span></p></td></tr><tr><td class="c12-21"><p class="c12-18 c12-14"><span class="c12-2">iGEM_39 (VR) 1:20</span></p></td><td class="c12-20"><p class="c12-0"><span>6μl</span></p></td></tr><tr><td class="c12-21"><p class="c12-18 c12-14"><span class="c12-2">dH</span><span class="c12-1">2</span><span class="c12-2">O</span></p></td><td class="c12-20"><p class="c12-0"><span>105μl</span></p></td></tr><tr><td class="c12-21"><p class="c12-14 c12-18"><span class="c12-2">Total</span></p></td><td class="c12-20"><p class="c12-0"><span>144μl</span></p></td></tr></tbody></table><p class="c12-15 c12-14"><span>→ addition of 24 μl MasterMix to each sample </span></p><p class="c12-9"><span>-</span><span> </span><span>PCR protocol: same as for colony-PCR</span></p><p class="c12-3"><span></span></p><p class="c12-3"><span></span></p><p class="c12-14"><span class="c12-2">Test restriction digest of Prom1</span><span class="c12-1">rev</span><span class="c12-2"> + part6.2, Prom3</span><span class="c12-1">rev</span><span class="c12-2"> + part 6.2 (C1 – C3 each)</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>of the plasmids prepped today, 2 μl were used for the digest, since the concentration could not be measured with NanoDrop</span></p><p class="c12-9"><span>-</span><span> </span><span>one reaction contained</span></p><p class="c12-3"><span></span></p><a href="#" name="30c7c869090768ae28bf86bdb7425dd700812cc5"></a><a href="#" name="3"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr><td class="c12-34"><p class="c12-7"><span class="c12-2">Component</span></p></td><td class="c12-42"><p class="c12-7"><span class="c12-2">Volume</span></p></td></tr><tr><td class="c12-34"><p class="c12-7"><span class="c12-2">plasmid</span></p></td><td class="c12-42"><p class="c12-7"><span>2μl</span></p></td></tr><tr><td class="c12-34"><p class="c12-7"><span class="c12-2">10x FD Green buffer</span></p></td><td class="c12-42"><p class="c12-7"><span>1μl</span></p></td></tr><tr><td class="c12-34"><p class="c12-7"><span class="c12-2">EcoRI FD</span></p></td><td class="c12-42"><p class="c12-7"><span>1μ</span></p></td></tr><tr><td class="c12-34"><p class="c12-7"><span class="c12-2">PstI FD</span></p></td><td class="c12-42"><p class="c12-7"><span>1μl</span></p></td></tr><tr><td class="c12-34"><p class="c12-7"><span class="c12-2">dH</span><span class="c12-1">2</span><span class="c12-2">O</span></p></td><td class="c12-42"><p class="c12-7"><span>5μl</span></p></td></tr><tr><td class="c12-34"><p class="c12-7"><span class="c12-2">Total</span></p></td><td class="c12-42"><p class="c12-7"><span>10μl</span></p></td></tr></tbody></table><p class="c12-7"><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>the plasmid was added to the tubes, then addition of 8 μl MasterMix; the MasterMix consisted of… </span></p><p class="c12-3"><span></span></p><a href="#" name="29b8402d804b319bd43369e3e12060c61cd63977"></a><a href="#" name="4"></a><table cellpadding="0" cellspacing="0" class="c12-27"><tbody><tr class="c12-61"><td class="c12-6"><p class="c12-7"><span class="c12-2">Component</span></p></td><td class="c12-45"><p class="c12-7"><span class="c12-2">Volume</span></p></td></tr><tr><td class="c12-6"><p class="c12-7"><span class="c12-2">10x FD Green buffer</span></p></td><td class="c12-45"><p class="c12-7"><span>7μl</span></p></td></tr><tr><td class="c12-6"><p class="c12-7"><span class="c12-2">EcoRI FD</span></p></td><td class="c12-45"><p class="c12-7"><span>7μl</span></p></td></tr><tr><td class="c12-6"><p class="c12-7"><span class="c12-2">PstI FD</span></p></td><td class="c12-45"><p class="c12-7"><span>7μl</span></p></td></tr><tr><td class="c12-6"><p class="c12-7"><span class="c12-2">dH</span><span class="c12-1">2</span><span class="c12-2">O</span></p></td><td class="c12-45"><p class="c12-7"><span>35μl</span></p></td></tr><tr><td class="c12-6"><p class="c12-7"><span class="c12-2">Total</span></p></td><td class="c12-45"><p class="c12-7"><span>56μl</span></p></td></tr></tbody></table><p class="c12-14"><span> </span></p><p class="c12-14"><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>incubation at 37 °C for 1 h</span></p><p class="c12-14 c12-43"><span> </span></p><p class="c12-14 c12-24"><span class="c12-2">Gel run:</span></p><p class="c12-14 c12-43"><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>1 % agarose-1xTAE gel</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 3 μl 2 log ladder</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 1 μl of uncut plasmid + 3 μl dH</span><span class="c12-10">2</span><span>O + 1 μl 5xLD</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 5 μl of RD reaction</span></p><p class="c12-9"><span>-</span><span> </span><span>run at 100 V</span></p><p class="c12-9"><span>-</span><span> </span><span>EtBr staining + destaining</span></p><p class="c12-9"><span>-</span><span> </span><span>UV detection</span></p><p class="c12-14"><span> </span><span class="c12-2">Loading:</span><span> Marker/Prom1</span><span class="c12-10">rev</span><span> + part6.2 C1 uncut/ Prom1</span><span class="c12-10">rev</span><span> + part6.2 C1 RD/ Prom1</span><span class="c12-10">rev</span><span> + part6.2 C2 uncut/ Prom1</span><span class="c12-10">rev</span><span> + part6.2 C2 RD/ Prom1</span><span class="c12-10">rev</span><span> + part6.2 C3 uncut/ Prom1</span><span class="c12-10">rev</span><span> + part6.2 C3 RD/ Prom3</span><span class="c12-10">rev</span><span> + part6.2 C1 uncut/ Prom3</span><span class="c12-10">rev</span><span> + part6.2 C1 RD/ Prom3</span><span class="c12-10">rev</span><span> + part6.2 C2 uncut/ Prom3</span><span class="c12-10">rev</span><span> + part6.2 C2 RD/ Prom3</span><span class="c12-10">rev</span><span> + part6.2 C3 uncut/ Prom3</span><span class="c12-10">rev</span><span> + part6.2 C3 RD/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/1/1d/Goe-19.08.13-RT-2.png" /><p class="c12-14"><span>part 6.2 Re-ligand: 35 bp (Promoter 3) + 8 bp (Scar between promoter and DarR operator) + 14 bp (operator) + 8 bp (Scar between operator and part 6 GFP) + 876 bp (part 6 GFP) = 941 → </span><span class="c12-2">ca. 940 bp should be visible in addition to pSB1C3 (2070 bp)</span></p><p class="c12-14"><span>part 6.2 C1 plasmid and Promoter1</span><span class="c12-10">rev</span><span>/Promoter3</span><span class="c12-10">rev</span><span>: 112 bp (rev. Promoter) + 8 bp (Scar between rev. promoter and promoter 3) + 35 bp (Promoter 3) + 8 bp (Scar between promoter and DarR operator) + 14 bp (operator) + 8 bp (Scar between operator and part 6 GFP) + 876 bp (part 6 GFP) = 1061 → </span><span class="c12-2">ca. 1060 bp should be visible in addition to pSB1C3 (2070 bp)</span></p><p class="c12-14"><span class="c12-2"> </span></p><p class="c12-14"><span class="c12-2 c12-55">Conclusion: </span></p><p class="c12-14"><span class="c12-55 c12-2">All clones seemed to be positive, since fragments have slightly more than 1 kb and ca. 2 kb. Sequencing of C1 and C2 of each.</span></p><p class="c12-8"><span class="c12-55 c12-2"></span></p><p class="c12-14"><span class="c12-2">Sequencing results from 16.8.13</span></p><p class="c12-14 c12-43"><span class="c12-2">Terminator</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 C1 and C3: </span><span>both clones contain the correct sequence of the terminator, but in reverse direction</span></p><p class="c12-9"><span class="c12-2">DarR</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 C1:</span></p><p class="c12-9"><span>-</span><span> </span><span>between NotI and PstI restriction site, an additional G is inserted</span></p><p class="c12-9"><span>-</span><span> </span><span>2</span><span class="c12-5">nd</span><span> base of 10</span><span class="c12-5">th</span><span> codon: A is missing</span></p><p class="c12-9"><span>-</span><span> → </span><span>don’t work with this clone!</span><span class="c12-2"> </span></p><p class="c12-9"><span class="c12-2">DarR</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 C4:</span></p><p class="c12-9"><span>-</span><span> </span><span>all mutations from the forward primer are present at correct site</span></p><p class="c12-9"><span>-</span><span> </span><span>no other mutations observed</span></p><p class="c12-9"><span>-</span><span> → </span><span> work with this clone</span></p><p class="c12-3"><span class="c12-2"></span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Restriction of Terminator</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 C1 for generating vector for ligation with DarR</span><span class="c12-1">re</span><span class="c12-10">v</span><span> </span></p><p class="c12-14 c12-43"><span>15 μl (ca. 1500 ng) plasmid Term</span><span class="c12-10">rev</span><span> C1 (from 16.8.13, 102.8 ng/μl)</span></p><p class="c12-14 c12-43"><span>4 μl SpeI FD</span></p><p class="c12-14 c12-43"><span>4 μl FD buffer 10x</span></p><p class="c12-14 c12-43"><span>17 μl dH</span><span class="c12-10">2</span><span>O</span></p><p class="c12-14 c12-43"><span>in total 40 μl</span></p><p class="c12-9"><span>-</span><span> </span><span>digest: 2 h at 37 °C</span></p><p class="c12-3"><span></span></p><p class="c12-14 c12-24"><span class="c12-2">Restriction of DarR</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 C4 for generating the insert for ligation with Term</span><span class="c12-1">rev</span><span class="c12-2"> C1 vector </span></p><p class="c12-14 c12-43"><span>6 μl (ca. 1500 ng) plasmid Term</span><span class="c12-10">rev</span><span> C1 (from 16.8.13, 282.5 ng/μl)</span></p><p class="c12-14 c12-43"><span>3μl XbaI FD</span></p><p class="c12-14 c12-43"><span>3 μl PstI FD</span></p><p class="c12-14 c12-43"><span>4 μl FD buffer 10x</span></p><p class="c12-14 c12-43"><span>24μl dH</span><span class="c12-10">2</span><span>O</span></p><p class="c12-14 c12-43"><span>in total 40 μl </span></p><p class="c12-9"><span>- </span><span>digest: 2 h at 37 °C</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Gel run for DarR</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 C4 and Terminator</span><span class="c12-1">rev</span><span class="c12-2"> + pSB1C3 C1 RDs and RBS</span><span class="c12-1">rev</span><span class="c12-2"> Test PCRs (all from today, see above)</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>1 % agarose-1xTAE gel</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 3 μl 2 log ladder</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 1 μl of uncut plasmid + 3 μl dH</span><span class="c12-10">2</span><span>O + 1 μl 5xLD</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 5 μl of RD reaction</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 5 μl PCR reaction (addition of 5 μl of 5xLD to each PCR sample)</span></p><p class="c12-9"><span>-</span><span> </span><span>run at 100 V</span></p><p class="c12-9"><span>-</span><span> </span><span>EtBr staining + destaining</span></p><p class="c12-9"><span>-</span><span> </span><span>UV detection</span></p><p class="c12-14"><span class="c12-2">Loading: </span><span>Marker/Terminator</span><span class="c12-10">rev</span><span> C1 uncut/Terminator</span><span class="c12-10">rev</span><span> + SpeI RD/DarR</span><span class="c12-10">rev</span><span> C4 uncut/DarR</span><span class="c12-10">rev</span><span> C4 + XbaI and PstI/RBS</span><span class="c12-10">rev</span><span> PCR: neg. control/RBS</span><span class="c12-10">rev</span><span> PCR: part 7 C1 plasmid/RBS</span><span class="c12-10">rev</span><span> PCR: RBS</span><span class="c12-10">rev</span><span> C1/RBS</span><span class="c12-10">rev</span><span> PCR: RBS</span><span class="c12-10">rev</span><span> C2/RBS</span><span class="c12-10">rev</span><span> PCR: RBS</span><span class="c12-10">rev</span><span> C3/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/0/05/Goe-19.08.13-RT-3.png" /><p class="c12-9"><span>-</span><span> </span><span>Term</span><span class="c12-10">rev</span><span> C1 is digested incompletely, but since the DarR</span><span class="c12-10">rev</span><span> insert won’t be dephosphorylated, one can dephosphorylate the incompletely digested vector → purifiy vector and digest with PstI</span></p><p class="c12-9"><span>-</span><span> </span><span>DarR</span><span class="c12-10">rev</span><span> C4 plasmid is digested completely → purify DarR</span><span class="c12-10">rev</span><span> fragment with gel extraction</span></p><p class="c12-9"><span>-</span><span> </span><span>PCR: the plasmids part 7 C1 and the RBS</span><span class="c12-10">rev</span><span> C1, C2 and C3 showed the expected products, but the negative control had a band at ca. 200 – 300 bp (I’ve no explanation for that: the water I used was the same for all other reactions! And the other material as well. Could be Bacillus spores, unclean PCR tubes, or my DNA…? XD → forget negative control (some people even don’t do no-template controls…), since expected products dominate/bands on neg. control seem not to be in lanes of all other samples → prepare C1 for sequencing</span></p><p class="c12-3"><span></span></p><p class="c12-3"><span></span></p><p class="c12-14"><span class="c12-2">Hybridization of RBS</span><span class="c12-1">rev</span><span class="c12-2">, Promoter1</span><span class="c12-1">rev</span><span class="c12-2"> and Promoter3</span><span class="c12-1">rev</span><span class="c12-2"> repeated </span></p><p class="c12-9"><span>-</span><span> </span><span>RBS</span><span class="c12-10">rev</span><span> must be cloned into Prom1</span><span class="c12-10">rev</span><span>/Prom3</span><span class="c12-10">rev</span><span> + part6.2 (from now on termed part 6.3A/B)</span></p><p class="c12-9"><span>-</span><span> </span><span>Promoter1</span><span class="c12-10">rev</span><span> and Promoter3</span><span class="c12-10">rev</span><span> must be cloned into shipping vector</span></p><p class="c12-9"><span>-</span><span> </span><span>hybridization performed as on 13.08.2013</span></p><p class="c12-9"><span>-</span><span> </span><span>samples stored at – 20 °C in DarR reporter system-box</span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Concentration measurement (NanoDrop worked again!) of plasmids purified today</span></p><img src="https://static.igem.org/mediawiki/2013/3/3e/Goe-19.08.13-RT-4.png" width="500" /><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">Preparation of samples for sequencing by SeqLab</span></p><p class="c12-9"><span>-</span><span> </span><span>sample no. 3: Promoter1</span><span class="c12-10">rev</span><span> + part6.2 C1 (part 6.3 A C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)</span></p><p class="c12-9"><span>-</span><span> </span><span>sample no. 4: Promoter1</span><span class="c12-10">rev</span><span> + part6.2 C2 (part 6.3 A C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)</span></p><p class="c12-9"><span>-</span><span> </span><span>sample no. 5: Promoter3</span><span class="c12-10">rev</span><span> + part6.2 C1 (part 6.3 B C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)</span></p><p class="c12-9"><span>-</span><span> </span><span>sample no. 6: Promoter3</span><span class="c12-10">rev</span><span> + part6.2 C2 (part 6.3 B C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)</span></p><p class="c12-9"><span>-</span><span> </span><span>sample no. 7: RBS</span><span class="c12-10">rev</span><span> + pSB1C3 C1 + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)</span></p><p class="c12-3"><span class="c12-2"></span></p><p class="c12-8"><span></span></p><p class="c12-14"><span class="c12-2">PCR clean up of Term</span><span class="c12-1">rev</span><span class="c12-2"> C1 + SpeI digest </span></p><p class="c12-9"><span>-</span><span> </span><span>with Qiagen PCR purification kit</span></p><p class="c12-9"><span>-</span><span> </span><span>500 μl PB buffer used</span></p><p class="c12-9"><span>-</span><span> </span><span>elution 1x with 30 μl HPLC water (pre-warmed), incubation at ca. 57°C for 2 min</span></p><p class="c12-9"><span>sample stored at – 20 °C in DarR reporter system box, marked with green </span></p><p class="c12-14 c12-24"><span class="c12-2">Gel ex of DarR</span><span class="c12-1">rev</span><span class="c12-2"> insert</span><span> </span></p><p class="c12-9"><span>-</span><span> </span><span>1%-agarose-1xTAE gel</span></p><p class="c12-9"><span>-</span><span> </span><span>addition of 9 μl 5x LD to 37 μl reaction</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of whole reaction on gel</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 3 μl marker</span></p><p class="c12-9"><span>-</span><span> </span><span>loading of 1 μl uncut DarR</span><span class="c12-10">rev</span><span> C4 plasmid + 3 μl dH</span><span class="c12-10">2</span><span>O + 1 μl 5xLD</span></p><p class="c12-9"><span>-</span><span> </span><span>run at 85 V</span></p><p class="c12-9"><span>-</span><span> </span><span>5 min staining with EtBr, short destaining in water, UV detection briefly, then gel ex under low UV light, tried to do gel ex, but signal was too weak, so again brief staining and destaining, then gel ex was possible</span></p><p class="c12-14"><span class="c12-2">Gel after gel ex:</span></p><img src="https://static.igem.org/mediawiki/2013/e/e1/Goe-19.08.13-RT-5.png" /><p class="c12-9"><span>-</span><span> </span><span>a bit was lost, but that’s ok…</span></p><p class="c12-9"><span>-</span><span> </span><span>gel pieces weighed and stored in DarR reporter system box at – 20 °C</span></p><p class="c12-3"><span></span></p><p class="c12-8"><span></span></p> | ||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | <p class="timeline-title goe-dac">Protein expression for crystallization of L. monocytogenes DAC domain</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt">Protein | ||
+ | expression for crystallization of L. monocytogenes DAC domain</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Preparation of 10l LB medium </span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Inoculation of 2x 50ml LB medium | ||
+ | containing ampicillin with 1013 cryo stock</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation o/n at 37°C with agitation</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><sub><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></sub><span lang="EN-US" style="font-size:12.0pt">Measurement of | ||
+ | OD<sub>600</sub></span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Inoculation of 10x 1l LB medium | ||
+ | containing ampicillin with culture having OD<sub>600</sub>=0,1</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation at 37°C until OD<sub>600</sub>=0,5-0,7</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Add each 1ml of 1M IPTG to each 1l of | ||
+ | culture</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation for 12-14h at 16°C with | ||
+ | agitation</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Measurement of OD<sub>600</sub> and | ||
+ | determine protein concentration and save samples for SDS-PAGE</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Transfer each 500ml of cultures into | ||
+ | centrifugation tubes and centrifuge for 10min at 5000rpm at 4°C</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Resuspend pellet in 20ml buffer W</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Pool solution of 2 pellets and | ||
+ | transfer protein solution into falcon tube</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Centrifugation for 15min at 5000rpm at | ||
+ | 4°C</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"; | ||
+ | color:red">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-US" style="font-size:12.0pt">Discard supernatant | ||
+ | and store pellet at -20°C</span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt"> </span></b></p> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
<div class="tlob" id="tl_0818"> | <div class="tlob" id="tl_0818"> | ||
<span class="date">18th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | <span class="date">18th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
<div class="cont"> | <div class="cont"> | ||
- | <p class="timeline-title">Riboswitch A Clones 1, 2, 4 and 5 inocculated into 4ml Cm cultures for minipreps tomorrow</p> | + | <p class="timeline-title goe-rt">Riboswitch A Clones 1, 2, 4 and 5 inocculated into 4ml Cm cultures for minipreps tomorrow</p> |
<div class="timeline-cont"> | <div class="timeline-cont"> | ||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | <p class="timeline-title goe-dac">Protein expression for crystallization of L. monocytogenes DAC domain</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt">Protein | ||
+ | expression for crystallization of L. monocytogenes DAC domain</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Preparation of 10l LB medium </span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Inoculation of 2x 50ml LB medium | ||
+ | containing ampicillin with 1013 cryo stock</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation o/n at 37°C with agitation</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><sub><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></sub><span lang="EN-US" style="font-size:12.0pt">Measurement of | ||
+ | OD<sub>600</sub></span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Inoculation of 10x 1l LB medium | ||
+ | containing ampicillin with culture having OD<sub>600</sub>=0,1</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation at 37°C until OD<sub>600</sub>=0,5-0,7</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Add each 1ml of 1M IPTG to each 1l of | ||
+ | culture</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation for 12-14h at 16°C with | ||
+ | agitation</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Measurement of OD<sub>600</sub> and | ||
+ | determine protein concentration and save samples for SDS-PAGE</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Transfer each 500ml of cultures into | ||
+ | centrifugation tubes and centrifuge for 10min at 5000rpm at 4°C</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Resuspend pellet in 20ml buffer W</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Pool solution of 2 pellets and | ||
+ | transfer protein solution into falcon tube</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Centrifugation for 15min at 5000rpm at | ||
+ | 4°C</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | ;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"; | ||
+ | color:red">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span><span lang="EN-US" style="font-size:12.0pt">Discard supernatant | ||
+ | and store pellet at -20°C</span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt"> </span></b></p> | ||
+ | |||
+ | |||
<div class="fbutton">Fold ↑</div> | <div class="fbutton">Fold ↑</div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> |
Latest revision as of 08:53, 17 October 2013
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Transformation of Promoter1rev/Promoter3rev + pSB1C3 , Colony PCR for Transformation of Promoter1rev/Promoter3rev + pSB1C3, Preparation of new Backup plates from yesterday’s ColonyPCRs/Inoculation...
Transformation of Promoter1rev/Promoter3rev + pSB1C3
- neg. control plates still negative
- pSB1C3 w/o insert control: 3 big clones on 50 μl-plate, many clones on rest-plate (small, big, medium in size)
- pSB1C3 + Promoter1rev: no clones on 50 μl-plate, 10 clones on rest-plate (small or medium size)
- pSB1C3 + Promoter3rev: no clones on 50 μl-plate, 6 clones on rest-plate (small or medium size)
→ Colony PCR to test for positive clones
→ plates stored at 4 °C, big fridge
Colony PCR for Transformation of Promoter1rev/Promoter3rev + pSB1C3
- as described previously (10.7.13)
- 1x RBSrev in pSB1C3 plasmid as additional control (plasmid prepped on 19.8.13, 211.6 ng/μl → 1:20 dilution → ca. 10 ng/μl), orange tube, termed RBS
- 1x medium size clone from w/o insert control plate picked (purple tube, termed R)
- 10x clones of Promoter1rev + pSB1C3 transformation picked (termed C1 – C10), blue tubes
- 6x clones of Promoter3rev + pSB1C3 transformation picked (termed C1 – C6), pink tubes
- preparation of mastermix for 20 reactions, primers: iGEM_38 (VF2) and iGEM_39 (VR)
- preparation of master plate (LBCm) → incubation over day at 37 °C
- 25 μl mastermix + 1 μl plasmid dilution or cells
- PCR protocol: same as on 10.7.13
- plates stored at 4 °C, big fridge
Preparation of new Backup plates from yesterday’s ColonyPCRs/Inoculation
- I wasn’t sure anymore, if I inoculated DarRrev-Termrev C1 – C5, part 6.4 B C1 – C5 and part 6.4A C1 – C8 correctly in the labeled tubes → streak-out of all clones for DarRrev-Termrev C1 – C5, part 6.4 B C1 – C5 and part 6.4A C1 – C8 on new LBCm plates
- in case of DarRrev-Termrev and part 6.4 B, the remaining clones on the master plates (that were not inoculated yesterday), were streaked out as well on the new master plate
- incubation over day at 37 °C, then storage at 4 °C, big fridge
MiniPlasmidPreparation of clones inoculated yesterday
- samples: DarRrev-Termrev C1 – C5, part 6.4 B C1 – C5 and part 6.4A C1 – C8
- with Nucleospin kit (Macherery-Nagel)
- 1st elution: 30 μl HPLC water (pre-warmed), incubation for 2 min at 50 °C
2nd elution: 22 μl HPLC water (pre-warmed), incubation for 2 min at 50 °C
- NanoDrop concentration measurement:
![](https://static.igem.org/mediawiki/2013/d/d2/Goe-23.08.13-RT-1.png)
Test Restriction Digest of DarRrev-Termrev C1 – C5
- 10 μl in total: 1 μl EcoRI FD + 1 μl PstI FD + 1 μl 10x FD Green buffer + 4 μl dH2O + 3 μl plasmid mix (ca. 200 – 300 ng plasmid)
- preparation of 6x Mastermix consisting of enzymes, buffer and water
- preparation of 3 μl plasmid-water Mix (plasmids prepped today, see above)
DarRrev-Termrev C1 → 3 μl plasmid (88.6 ng/μl)
DarRrev-Termrev C2 → 2 μl plasmid (160.2 ng/μl) + 1 μl dH2O
DarRrev-Termrev C1 → 1 μl plasmid (229.7 ng/μl) + 2 μl dH2O
DarRrev-Termrev C1 → 3 μl plasmid (88.5 ng/μl)
DarRrev-Termrev C1 → 2μl plasmid (107.3 ng/μl) + 1 μl dH2O
- addition of 7 μl Mastermix
- incubation at 37 °C for 1 h
Gel run for Test RD of DarRrev-Termrev C1 – C5, plasmids from today’s MiniPrep and Colony PCR for Transformation of Promoter1rev/Promoter3rev + pSB1C3
- 3x 1%-agarose-1xTAE gel
- addition of 5 μl 5xLD to 25 μl Colony PCR reaction and loading of 5 μl PCR reaction supplied with 5xLD (during loading, some sample Colony PCR Promoter3rev+pSB1C3 C4 was spilled over the well…)
- loading of 1 μl uncut DarRrev-Termrev C1 – C5 plasmid + 1 μl 5x LD + 3 μl dH2O; loading of 5 μl RD reaction mix
- loading of 1 μl uncut part 6.4 B C1 – C5 and part 6.4A C1 – C8 plasmid + 1 μl 5x LD + 3 μl dH2O
- loading of 3 μl 2 log ladder as a marker
- run at 100 V
- EtBr staining + destaining in water
- UV detection
Gel 1:
Loading: Marker/uncut DarRrev-Termrev C1/ RD DarRrev-Termrev C1/ uncut DarRrev-Termrev C2/ RD DarRrev-Termrev C2/ uncut DarRrev-Termrev C3/ RD DarRrev-Termrev C3/ uncut DarRrev-Termrev C4/ RD DarRrev-Termrev C4/ uncut DarRrev-Termrev C5/ RD DarRrev-Termrev C5/part 6.4 B C1 plasmid/part 6.4 B C2 plasmid/part 6.4 B C3 plasmid/part 6.4 B C4 plasmid/Marker
![](https://static.igem.org/mediawiki/2013/e/ee/Goe-23.08.13-RT-2.png)
expected sizes for DarRrev-Termrev plasmids: 630 bp (DarRrev) + 130 bp (Terminatorrev) = 760 bp and 2070 bp from pSB1C3 backbone
→ all DarRrev-Termrev clones are positive → sequencing of C2 and C3 by SeqLab (because these clones have highest plasmid concentration)
→ all purified part 6.4 B plasmids look normal except for plasmid part 6.4 B C1 and C3 → sequencing of C2 and C5 by SeqLab (because these clones have highest plasmid concentration and because their plasmids seem to be ok)
Gel 2:
Loading: Marker/part 6.4 B C5 plasmid/ part 6.4A C1 plasmid/ part 6.4A C2 plasmid/ part 6.4A C3 plasmid/ part 6.4A C4 plasmid/ part 6.4A C5 plasmid/ part 6.4A C6 plasmid/ part 6.4A C7 plasmid/ part 6.4A C8 plasmid/Colony PCR RBSrev+pSB1C3 plasmid/ Colony PCR Re-ligand/Colony PCR Promoter1rev+pSB1C3 C1/ Colony PCR Promoter1rev+pSB1C3 C2/ Colony PCR Promoter1rev+pSB1C3 C3/Marker
![](https://static.igem.org/mediawiki/2013/3/30/Goe-23.08.13-RT-3.png)
expected sizes for Promoter1rev and Promoter3rev Colony PCR products: 310 bp (VR, VF2) + 112 bp Promoter1/3rev = 422 bp
expected sizes for RBSrev+pSB1C3 and Re-ligand: 310 bp (VR, VF2) + 12 bp (RBSrev) = 322 bp
→ all purified part 6.4 B/A plasmids look normal → sequencing of part 6.4 A C2, C3, C5 and C8 by SeqLab (because these clones have highest plasmid concentration)
→ Colony PCR: for RBSrev+pSB1C3 and Re-ligand, the expected band was observed, band at same bp for Promoter1rev+pSB1C3 C1 – C3 (negative clones)
Gel3:
Loading: Marker/ Colony PCR RBSrev+pSB1C3 plasmid/ Colony PCR Promoter1rev+pSB1C3 C4/ Colony PCR Promoter1rev+pSB1C3 C5/ Colony PCR Promoter1rev+pSB1C3 C6/ Colony PCR Promoter1rev+pSB1C3 C7/ Colony PCR Promoter1rev+pSB1C3 C8/ Colony PCR Promoter1rev+pSB1C3 C9/ Colony PCR Promoter1rev+pSB1C3 C10/ Colony PCR Promoter3rev+pSB1C3 C1/ Colony PCR Promoter3rev+pSB1C3 C2/ Colony PCR Promoter3rev+pSB1C3 C3/ Colony PCR Promoter3rev+pSB1C3 C4/ Colony PCR Promoter3rev+pSB1C3 C5/ Colony PCR Promoter3rev+pSB1C3 C6/Marker
![](https://static.igem.org/mediawiki/2013/c/ce/Goe-23.08.13-RT-4.png)
expected sizes for Promoter1rev and Promoter3rev Colony PCR products: 310 bp (VR, VF2) + 112 bp Promoter1/3rev = 422 bp
expected sizes for RBSrev+pSB1C3 and Re-ligand: 310 bp (VR, VF2) + 12 bp (RBSrev) = 322 bp
→ Colony PCR: for RBSrev+pSB1C3, the expected band was observed, band at same bp for Promoter1rev+pSB1C3 C5 and for Promoter3rev+pSB1C3 C1 – C4 and C6 (negative clones); band at ca. 400 bp for romoter1rev+pSB1C3 C4 and Promoter3rev+pSB1C3 C5 (= positive clones)→ on Monday: inoculation of these clones for MiniPrep, test RD and sequencing
Preparations of samples for sequencing by SeqLab
9 – DarRrev-Termrev C2 + VF2
10 – DarRrev-Termrev C2 + VR
11 – DarRrev-Termrev C3 + VF2
12 - DarRrev-Termrev C3 + VR
13 – part6.4 B C2 + VF2
14 – part 6.4 B C5 + VF2
15 – part 6.4 A C2 + VF2
16 – part 6.4 A C3 + VF2
17- part 6.4 A C5 + VF2
18 – part 6.4 A C8 + VF2
- for all samples: 6 μl plasmid + 1 μl iGEM_38 1:5 or iGEM_39 1:5
Cryostocks and Minipreps of Ribo A C4 and C5 inocculated by Nina yesterday
Following clones were frozen away prepped:
Samples | Procedures done |
C4 3 | Cryostock and Mini |
C4 4 | Cryostock and Mini |
C4 6 | Cryostock and Mini |
C4 7 | Cryostock |
C4 8 | Cryostock and Mini |
C5 2 | Cryostock and Mini |
C5 3 | Cryostock and Mini |
C5 6 | Cryostock and Mini |
C5 8 | Cryostock and Mini |
- C4 7 was dropped during the miniprep
Nanodrop:
All ca. 400-500ng/µl , good ratio, exact measurements on the vials
Sequencing by SeqLab:
1: Ribo A C4 6 VF
2: Ribo A C4 6 VR
3: Ribo A C4 8 VF
4: Ribo A C4 8 VR
5: Ribo A C5 6 VF
6: Ribo A C5 6 VR
7: Ribo A C5 8 VF
8: Ribo A C5 8 VR
Inoculation of 10ml cultures of Ribo A C4 7 and 8, the DAC cells and an empty vector as control (names to be added later) for an experiment with plates containing either the reporter system or the DAC producing cells in the agar. Clones were inoculated by Katrin G. yesterday.
Our Ribo A clones didn´t grow at all, the other two cultures were harvested at OD600=2, washed and frozen away at -80°C in Glycerin as follows:
→ harvest 4x 2ml of the culture in 2ml eppis
→ centrifuge down
→ discard supernatant
→ resuspend cells in 100µl LB medium
→ transfer the resuspended cells into a 2ml screw-cap-eppi
→ add 200µl Glycerin
→ Vortex and freeze at -80°C
SDS Page of dialysis and elution samples
SDS Page of dialysis and elution samples
- Mix 15µl of samples with 5µl Pab and 5µl buffer W
- Incubation for 10min at 93°C
- Mix each sample with 4µl protein marker
- Load whole volume on 15% SDS-gel
- Run for 5min at 90V and 1h at 120V
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Colony PCR for DarRrev-Termrev transformation , Colony PCR for part6.3 B + RBSrev transformation, Gel run for Colony PCR for DarRrev-Termrev transformation, Sequencing of part6.3 B C1, C2 and C3 plasmids by G2L, Plates of part6.3 A + RBSrev transformation...
Transformation from 21.8.13
- neg. control: no clones on 50 μl-plate, no clones on rest-plate
- Termrev vector w/o insert: no clones on 50 μl-plate, few clones on rest-plate
- Termrev vector + DarRrev insert: few clones on 50 μl-plate, many clones on rest-plate
- part 6.3 A vector w/o insert: no clones on 50 μl-plate, 1 clone on rest-plate
- part 6.3 A vector + RBSrev insert: no clones on 50 μl-plate, few clones on rest-plate
- part 6.3 B vector w/o insert: no clones on 50 μl-plate, no clones on rest-plate
- part 6.3 B vector + RBSrev insert: no clones on 50 μl-plate, some clones on rest-plate
- pSB1C3 vector w/o insert: 1 or 2 clones on 50 μl-plate, several clones on rest-plate
- pSB1C3 vector + Promoter1rev insert: no clones on 50 μl-plate, no clones on rest-plate
- pSB1C3 vector + Promoter3rev insert: no clones on 50 μl-plate, few clones on rest-plate
→ colony PCR for DarRrev-Termrev transformation
→ further incubation of all other plates, until clones are bigger/grown
Colony PCR for DarRrev-Termrev transformation
- as described previously (10.7.13)
- 1x Termrev C1 plasmid as additional control (plasmid prepped on 16.8.13, 102.8 ng/μl → 1:10 dilution → ca. 10 ng/μl), blue tube (termed T)
- 1x Re-ligand (termed R), pink tube
- 12x DarRrev-Termrev clones (termed C1 – C12), purple tubes
- preparation of mastermix for 15 reactions, primers: iGEM_38 (VF2) and iGEM_39 (VR)
- preparation of master plate (LBCm) → incubation over day at 37 °C
- 25 μl mastermix + 1 μl plasmid dilution or cells
- PCR protocol: same as on 10.7.13
- plates stored at 4 °C, big fridge
Plates for part 1 – 4 C1 – C3
- plates taken out, since all clones grew
- plates put to 4 °C, big fridge
Colony PCR for part6.3 B + RBSrev transformation
- after some additional incubation time
- as described previously (10.7.13)
- 1x part6.3 B C2 plasmid as additional control (plasmid prepped on 19.8.13, 220.3 ng/μl → 1:20 dilution → ca. 11 ng/μl), purple tube, termed 6.3 B
- neg. control plates still without colonies
- still no religands → performing dephosphorylation longer than actually necessary seems to be helpful (see 20.8.13)
- 1 or 2 clones on 50 μl plate, many clones on rest-plate: 13x clones picked (termed C1 – C13), green tubes
- preparation of mastermix for 15 reactions, primers: iGEM_38 (VF2) and iGEM_39 (VR)
- preparation of master plate (LBCm) → incubation over day at 37 °C
- 25 μl mastermix + 1 μl plasmid dilution or cells
- PCR protocol: same as on 10.7.13
- plates stored at 4 °C, big fridge
→ 12 bp (size of RBSrev) difference between positive and negative clones, one would never see in a gel… PCR thrown away…
Gel run for Colony PCR for DarRrev-Termrev transformation
- 1%-agarose-1xTAE gel
- addition of 5 μl 5xLD to 25 μl PCR reaction
- loading of 3 μl 2 log ladder as a marker
- loading of 5 μl PCR reaction supplied with 5xLD
- run at 100 V
- EtBr staining + destaining in water
- UV detection
Loading: Marker/Termrev plasmid/Re-ligand/ DarRrev-Termrev C1/ DarRrev-Termrev C2/ DarRrev-Termrev C3/ DarRrev-Termrev C4/ DarRrev-Termrev C5/ DarRrev-Termrev C6/ DarRrev-Termrev C7/ DarRrev-Termrev C8/ DarRrev-Termrev C9/ DarRrev-Termrev C10/ DarRrev-Termrev C11/ DarRrev-Termrev C12/Marker
![](https://static.igem.org/mediawiki/2013/6/6f/Goe-22.08.13-RT-1.png)
- expected product sizes
negative clone: 310 bp (VR, VF2) + 130 bp (Terminatorrev) = 440 bp
positive clone: 310 bp (VR, VF2) + 130 bp (Terminatorrev) + 630 bp (DarRrev) = 1070 bp
→ Religand plasmid corresponds to Termrev plasmid, as expected
→ all DarRrev-Termrev clones appear to be positive → inoculate 3 clones for MiniPrep tomorrow
- PCR reactions stored at 4 °C, small fridge
Sequencing of part6.3 B C1, C2 and C3 plasmids by G2L
sequencing by SeqLab failed; for both clones, sequence stopped at same region: possible reason: secondary structures formed during sequencing
5 μl total reaction: ca. 300 ng plasmid + 10 % DMSO (to prevent secondary structures) + 1 μl primer iGEM_38, 1:20 diluted
Preparation of the following samples
- ppra_1: 2 μl plasmid prepped on 19.8.13 (268.4 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water
- ppra_2: 2 μl plasmid prepped on 19.8.13 (220.3 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water
- ppra_3: 2 μl plasmid prepped on 19.8.13 (247.2 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water
- ppra_4: 1 μl plasmid prepped on 21.8.13 (305.4 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water
- ppra_5: 1 μl plasmid prepped on 21.8.13 (332.1 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water
- ppra_6: 1 μl plasmid prepped on 21.8.13 (328.8 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water
prior to sequencing, the samples have to be denatured → remark was entered into excel-sheet
Plates of part6.3 A + RBSrev transformation
- still no clones on neg. control
- 3 clones on w/o insert plate
- 8 clones on w/ insert plate
Inoculation of DarRrev-Termrev clones, part6.3 B + RBSrev(= part 6.4 B) and part 6.3 A + RBSrev(= part 6.4 A) clones for MiniPrep
- inoculation of
DarRrev-Termrev clones C1 – C5→ Test restriction digest
B clones C1 – C5 (no re-ligands)→ sequencing
part6.4 A clones C1 – C8 (3 re-ligands and 8 clones on WITH-insert plate→ sequencing
- in 4 ml LBCm
- incubation at 37 °C ON
Run the colony PCR from yesterday on a gel
Gel doc:
Wells: M/Ribo A C4 clone 1/2/3/4/5/6/7/8/9/10/blank/religant from control plate w/o insert
![](https://static.igem.org/mediawiki/2013/6/6a/Goe-22.08.13-RT-2.png)
Wells: M/Ribo A C5 clone 1/2/3/4/5/6/7/8/9/10/blank/religant from control plate w/o insert
![](https://static.igem.org/mediawiki/2013/7/78/Goe-22.08.13-RT-3.png)
→ all clones with the high band should contain the Ribo A insert
Following clones were choosen to prepare a miniprep:
Ribo A C4 clones 3/4/6/7/8 (all others didn’t grew or showed wrong bands)
Ribo A C5 clones 2/3/6/8 (all others didn’t grew or showed wrong bands)
Miniprep of the DAC team Lm cryostock colony
Nanodrop:
Sample | ng/µl | A260nm/A280nm | A260nm/A230nm |
C4 3 | 15.8 | 0.82 | 0.18 |
C4 4 | 105.8 | 1.92 | 0.89 |
C4 6 | 46.8 | 1.44 | 0.41 |
C4 7 | 223.6 | 1.69 | 0.93 |
C4 8 | 85.2 | 1.53 | 0.73 |
C5 2 | 23.3 | 1.74 | 2.27 |
C5 3 | 56.0 | 1.97 | 0.87 |
C5 6 | 154.9 | 1.56 | 0.71 |
C5 8 | -0.1 | -0.06 | -0.05 |
DAC Lm | 36.5 | 1.75 | 1.91 |
Second elute C5 2 | 17.5 | 1.46 | 0.65 |
Second elute DAC Lm | 51.1 | 1.83 | 2.41 |
Second measurement C5 8 | 3.2 | 1.37 | 0.37 |
Stored in to do box
Following plasmids were choosen for sequencing:
Seq number | Plasmid + primer |
1 | C4 clone 7 for |
2 | C4 clone 7 rev |
3 | C4 clone 6 for |
4 | C4 clone 6 rev |
5 | C5 clone 3 for |
6 | C5 clone 3 rev |
7 | C5 clone 6 for |
8 | C5 clone 6 rev |
Again inocculation of all preped Ribo A C4 and C5 clones that were already prepped, for preparation of Cryostocks and a new miniprep, as the old one showed smears
Katrin Gunka
Preparation of plates with Lb medium also containing the DAC team Lm stain. One plate just like this, another one also containing
RT-PCR
RT-PCR
· Another gel to check the digestion from the day before and to check the newly ordered primers for ydaO and ktrA.
· The gel looked fine, so the RT-PCR can get started
Concentrations digested RNA
· digestion 20.08.13
o wt: 76 ng/µl
o 44: 73 ng/µl
o 46: 83 ng/µl
o wt*: 87 ng/µl
o 44*: 73 ng/µl
o 46*: 69 ng/µl
· digestion 21.08.13
o wt: 63 ng/µl
o 44: 64,5 ng/µl
o 46: 72 ng/µl
Results RT-PCR
· no significant differences. All genes seem to be downregulated in both strains (1344 = much c-di-AM; 1346 = little c-di-AMP)
Ø for better differences a new RT-PCR with differrent conditions are tested (medium: low and high phosphate medium)
These results show us, that there is no real difference between the strains, because all genes are downregulated.ptsH, hrcA and groEL, served as control. We are mainly interested in ydaO and ktrA. YdaO is the gene behind the riboswitch, ktrA is a phosphate channel. Due to the fact that there is no difference between 1344 and 1346 compared to the wildtype in CSE 0,5% Glucose medium, we did the exact same setup only in low & high phosphate medium (see below).
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Plasmid Mini Prep, Transformation of ligations prepared yesterday, observation of clones of Ribo A C4 and C5 CFP under UV light...
CryoStocks
- preparation of DMSO cryostocks of all inoculated clones (according to protocol described previously)
Plasmid Mini Prep
- MiniPrep was done using the culture volume that remained after preparing the cryostocks (see above)
- kit: Nucleospin, Macherey-Nagel
- cells were harvested by centrifuging at 13000 g, 1 min
- elution: 1x 30 μl HPLC water (pre-warmed), incubation for 3 min at 50 °C
- NanoDrop concentration measurement:
![](https://static.igem.org/mediawiki/2013/6/6c/Goe-21.08.13-RT-1.png)
Transformation of ligations prepared yesterday
- according to protocol in methods folder
- with XL1-Blue comp. cells
- neg. control was done
- when adding the Promoter1rev + pSB1C3 ligation mix to the comp. cells, a drop of the comp. cells was spilled…
- 500 μl were removed after centrifuging
- plating on LBCm plates
- incubation at 37 °C
Gel run after MiniPrep to check if plasmids are clean
- 1 %-agarose-1xTAE gel from yesterday (prepared by Jan)
- loading of 3 μl 2 log ladder
- loading of 1 μl plasmid + 1 μl 5xLD + 3 μl dH2O
- run at 100 V
- EtBr staining + destaining in water
- UV detection
loading: Marker/Termrev C1/DarRrev C4/ RBSrev C1/part6.3 A C1/part6.3 B C1/ part6.3 B C2/ part6.3 B C3
![](https://static.igem.org/mediawiki/2013/f/fb/Goe-21.08.13-RT-2.png)
→ all plasmids are purified from chrom.DNA and look normal
→ samples stored in DarR reporter system boxRe-streak of part 1 – 4 Clones 1 – 3 on LBAmp
- plates from 6.6.13 (stored in fridge…. at 4 °C….)
- bacteria were quite dry, so I scratched some cell material off and streaked it out on new plates
- incubation ON at 37 °C
- let’s hope they grow…
Preparation of new LBCm plates
- 2x500 ml
- 35 μg/ml Cm
- stored at 4 °C, big fridge
observation of clones of Ribo A C4 and C5 CFP under UV light
(all clones with YFP don’t glow under UV light)
Pictures:
Ribo A C4 + CFP UV light
![](https://static.igem.org/mediawiki/2013/3/3c/Goe-21.08.13-RT-3.png)
Ribo A C4 + CFP transmitted light
![](https://static.igem.org/mediawiki/2013/e/e1/Goe-21.08.13-RT-4.png)
Ribo A C5 + CFP UV light
![](https://static.igem.org/mediawiki/2013/f/f3/Goe-21.08.13-RT-5.png)
Ribo A C5 + CFP transmiited light
![](https://static.igem.org/mediawiki/2013/8/82/Goe-21.08.13-RT-6.png)
Control w/o insert UV light
![](https://static.igem.org/mediawiki/2013/0/00/Goe-21.08.13-RT-7.png)
Control w/o insert transmitted light
![](https://static.igem.org/mediawiki/2013/9/90/Goe-21.08.13-RT-8.png)
Preparation of a Masterplate of 10 clones from plate with C4 and C5 CFP
Inocculation of 10 clones from plate with C4 and C5 CFP in LB medium with Cm
Colony PCR of the inoculated clones and a control w/o insert CFP
Component | Volume |
Taq | 1μl |
10x Taq buffer | 2,8μl |
dNTP mix | 1μl |
iGEM_38 (VF2) 1:20 | 1μl |
iGEM_39 (VR) 1:20 | 1μl |
dH2O | 18,8μl |
Total | 25μl |
DNAse I digestion
DNAse I digestion
· this digestion is necessary to get rid of DNA contamination
· use PCR tubes
25 µl sample
· 2.5 µg RNA
· 2.5 µl 10 x buffer
· 5 µl DNAse I
· x µl RNAse free water
30 min 37°C in the PCR cycler
Stop the reaction with 2.5 µl EDTA for 10 min at 65°C (also in the cycler)
Control PCR on digestion
PCR Scheme (50 µl)
· 10 µl HF buffer
· 2 µl dNTPs Mix
· 4 µl Primer Mix (here rpsE)
· 1 µl RNA
· 0,5 µl PhuS
· 32 µl RNAse free H20.
PCR Programm
· 98°C 2 min
· 98°C 35 sec
· 52°C 35 sec
· 72°C 45 sec
· 72°C 10 min
· 15°C hold
· run a gel to check if everything went correctly
In this case we did this digestion with the wt, GP1344 and GP1346. Additionally we used a NTC and cDNA control.
Ø The digestion worked fine. No DNA detectable in our samples, only in the cDNA
Ø Determine RNA concentration
Ø This failed
We did the digestion again. This time we used 6 samples in total, two samples of each strain. IN the first three samples we used 5 µl DNAse I according to protocol, in the other three samples we only used 2.5 µl DNAse I.
Stored the samples over night at -20°C.
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Gel extraction of DarRrev insert , Restriction digest of Termrev with PstI FD, Restriction digest of RBSrev C1 and part6.3 A and B (C2 each) with EcoRI FD, AP treatment of Termrev vector for ligation with DarRrev insert , Restriction Digest of all vectors digested with EcoRI FD today, ligation.....
Transformation of yesterday´s ligation
Gel extraction of DarRrev insert
- with Qiagen PCR gel/PCR purification kit
- since DarRrev insert has > 600 bp, no isopropanol was added/only QG buffer used
- only 1 column used since <400 mg gel
- elution 1x with 30 μl of pre-heated HPLC water, incubation at 50 °C for 2 min
- NanoDrop concentration measurement
sample | ng/μl | A260nm/A280nm | A260nm/A230nm |
DarRrev insert X+P | 14.8 | 1.53 | 0.12 |
Restriction digest of Termrev with PstI FD
- DNA: Termrev C1 plasmid restricted with SpeI and purified yesterday
- digest:
Component | Volume |
plasmid | 30μl (volume determined with pipet) |
10x FD Green buffer | 4μl |
PstI FD | 4μl |
dH2O | 2μl |
Total | 40μl |
- digest for 2 h at 37 °C
Restriction digest of RBSrev C1 and part6.3 A and B (C2 each) with EcoRI FD
- DNA: plasmids purified yesterday
- digest for all reactions (pipette individually, no mastermix):
Component | Volume |
plasmid | 8μl (since each plasmid had > 200 ng/μl) |
10x FD Green buffer | 4μl |
Eco I FD | 4μl |
dH2O | 24μl |
Total | 40μ |
- digest for 2 h at 37 °C
Sequencing
results RBSrev C1 and part6.3 A and B (C1 and C2 each)
- part 6.3 A C1 (sample 3) – sequence as expected (no mutations)
- part 6.3 A C2 (sample 4) – a G at position 52 of Hybridization oligo is missing → digest sample (see above) thrown away, new digest (exactly as described above) using clone 1
- part 6.3 B C1 (sample 5) – sequencing did not work, only first part of oligo in vector sequenced → was ok
- part 6.3 B C2 (sample 6) – sequencing did not work, only first part of oligo in vector sequenced → was ok → further digest
- RBSrev in pSB1C3 C1 (sample 7) – sequence as expected
Loading:
Marker/uncut Termrev C1/RD Termrev C1/uncut RBSrev C1/RD RBSrev C1/uncut part6.3 A C1/RD part6.3 A C1/uncut part6.3 B C2/RD part6.3 B C2/Marker
![](https://static.igem.org/mediawiki/2013/b/b3/Goe-20.08.13-RT-1.png)
- all digests are only partial, but most of plasmid is cut → purify digests
New sizes of Riboswitch PCR products
I checked again the alignments of the riboswitch PCR products, and the sizes I entered into the journal on 24.6.13 are partially wrong.
The correct sizes are:
| Primers | approx. PCR product size (w/o prefix/suffix) | approx. PCR product size (w/ prefix/suffix) |
Riboswitch with native Promoter and RBS | iGEM_42 iGEM_43 | 460 bp | 500 bp |
Riboswitch with native Promoter | iGEM_42 iGEM_41 | 315 bp | 355 bp |
Riboswitch only | iGEM_40 iGEM_41 | 230 bp | 270 bp |
Riboswitch with native RBS | iGEM_40 iGEM_43 | 375 bp | 415 bp |
Inoculation of clones for CryoStocks
inoculation of
- RBSrev + pSB1C3 C1
- part 6.3 A C1
- part 6.3 B C1, C2, C3
- Termrev + pSB1C3 C1
- DarRrev + pSB1C3 C4
→ from master plates, inoculation in 4 ml LBCm
incubation ON at 37 °C, 200 – 210 rpm
Purification of vectors digested today
- samples: RBSrev + pSB1C3 RD, part 6.3 A C1 and part 6.3 B C2 RD, Termrev + pSB1C3 RD
- with Qiagen PCR clean-up kit
- addition of 500 μl PB buffer to each reaction
- elution with 30 μl pre-warmend HPLC water, incubation at 50 °C for 2 min
AP treatment of Termrev vector for ligation with DarRrev insert
- this is silly… I could have done it before purification, then I would have lost less vector…
- addition of 1 μl AP, 4 μl AP buffer 10x, 8 μl dH2O to 27 μl purified vector
- incubation for 1 h at 37 °C (PstI makes 3’ overhang)
Restriction Digest of all vectors digested with EcoRI FD today
- samples: RBSrev + pSB1C3 RD, part 6.3 A C1 and part 6.3 B C2 RD
- all samples had a volume of 30 μl after clean-up, so addition of 2 μl dH2O and 4 μl 10x FD buffer
- addition of 4 μl SpeI FD to RBSrev + pSB1C3 RD sample, and 4μl XbaI FD to part 6.3 A C1 and part 6.3 B C2 RD samples
- for all reactions: total volume of 40 μl
- part 6.3 A C1 and part 6.3 B C2 RD digests incubated at 37 °C for 1 h (then dephosphorylation)
- RBSrev + pSB1C3 RD digest incubated at 37 °C for 2 h (no dephosphorylation, since Promoter hybridization oligos are dephosphorylated!)
AP treatment of part 6.3 A C1 and B C2
- 40 μl reaction + 5 μl 10x AP buffer + 2 μl AP + 3 μl dH2O
- incubation for ca. 50 min at 37 °C (although only 15 min needed, since XbaI and EcoRI generate 5’ overhangs. But dephosphorylation often incomplete…)
Gel run of digested and AP treated part 6.3 A C1 and BC2 and RBSrev digested with SpeI and EcoRI
- 1%-agarose-1xTAE gel
- loading of 3 μl 2 log ladder
- loading of 4 μl of dephosphorylated reactions + 1 μl 5xLD
- loading of 3 μl RBSrev digest + 1 μl dH2O + 1μl 5xLD
- run at 100 V
- EtBr staining + destaining in water
- UV detection
Loading:
Marker/ uncut RBSrev C1/RD RBSrev C1/uncut part6.3 A C1/RD + AP part6.3 A C1/uncut part6.3 B C2/RD + AP part6.3 B C2/Marker
![](https://static.igem.org/mediawiki/2013/7/79/Goe-20.08.13-RT-2.png)
- no over-digest; digests still incomplete, but since AP treatment of at least part 6.3 A and B vector, this should not matter; in case of RBSrev ligation, re-ligands could occur
Purification of digested and dephosphorylated samples
- with Qiagen PCR purification kit
- addition of 500 μl PB buffer to each sample
- elution 1x with 30 μl HPLC water (pre-warmed), incubation for 2 min at 50 °C
- NanoDrop concentration measurement
![](https://static.igem.org/mediawiki/2013/d/d4/Goe-20.08.13-RT-3.png)
Ligation
Ligation of
a) DarRrev insert + Termrev vector → Termrev-DarRrev in pSB1C3
b) RBSrev hybridization oligo + part6.3 A/part 6.3 B → part 6.4 A/part 6.4 B (RBSrev-Promoter1/3rev in part 6.2)
c) Promoter1/3rev + pSB1C3 derived from RBSrev in pSB1C3 digest → Promoter1/3rev in pSB1C3
Pipetting scheme
a) DarRrev insert + Termrev vector → Termrev-DarRrev in pSB1C3
Uni D’dorf ligation calculator: for a ratio vector:insert = 1:3, one has to use 50 ng of a 2200 bp (= 2070 of pSB1C3 + 130 bp of terminator) vector and 44 ng insert of 650 bp → approximate amounts used in this ligation.
Component | With DarRrev insert | Without insert control |
Termrev vector (purified today, 19.9 ng/µl) | 3 µl | 3 µl |
DarRrev insert (purified today, 14.8 ng/µl) or dH2O for w/o insert control | 3 µl | 3 µl |
T4 ligase (ThermoScientific) | 1 µl | 1 µl |
10x T4 ligase buffer (ThermoScientific) | 1 µl | 1 µl |
dH2O | 2 µl | 2 µl |
Total | 10 µl | 10 µl |
b) RBSrev hybridization oligo + part6.3 A/part 6.3 B → part 6.4 A/part 6.4 B (RBSrev-Promoter1/3rev in part 6.2)
Component | With RBSrev insert | Without insert control |
Part 6.3 A vector (purified today, 30.5 ng/µl) | 2 µl | 2 µl |
RBSrev hybridization oligo (prepared on 19.8.13) or dH2O for w/o insert control | 8 µl | 8 µl |
T4 ligase (ThermoScientific) | 2 µl | 2 µl |
10x T4 ligase buffer (ThermoScientific) | 2 µl | 2 µl |
dH2O | 6 µl | 6 µl |
Total | 20 µl | 20 µl |
Component | With RBSrev insert | Without insert control |
Part 6.3 B vector (purified today, 33.6 ng/µl) | 2 µl | 2 µl |
RBSrev hybridization oligo (prepared on 19.8.13) or dH2O for w/o insert control | 8 µl | 8 µl |
T4 ligase (ThermoScientific) | 2 µl | 2 µl |
10x T4 ligase buffer (ThermoScientific) | 2 µl | 2 µl |
dH2O | 6 µl | 6 µl |
Total | 20 µl | 20 µl |
c) Promoter1/3rev + pSB1C3 derived from RBSrev in pSB1C3 digest → Promoter1/3rev in pSB1C3
Component | With Promoter1rev insert | With Promoter3rev insert | Without insert control |
psB1C3 vector (derived from RBSrev C1 plasmid, purified today, 29.9 ng/µl) | 2 µl | 2 µl | 2 µl |
Promoter1/3rev hybridization oligo (prepared on 19.8.13) or dH2O for w/o insert control | 10 µl | 10 µl | 10 µl |
T4 ligase (ThermoScientific) | 2 µl | 2 µl | 2 µl |
10x T4 ligase buffer (ThermoScientific) | 2 µl | 2 µl | 2 µl |
dH2O | 4 µl | 4 µl | 4 µl |
Total | 20 µl | 20 µl | 20 µl |
- All reaction were pipetted individually/no mastermix
- Incubation ON at 16 °C (cold room heat block)
DNAse I digestion
DNAse I digestion
· this digestion is necessary to get rid of DNA contamination
· use PCR tubes
25 µl sample
· 2.5 µg RNA
· 2.5 µl 10 x buffer
· 5 µl DNAse I
· x µl RNAse free water
30 min 37°C in the PCR cycler
Stop the reaction with 2.5 µl EDTA for 10 min at 65°C (also in the cycler)
Control PCR on digestion
PCR Scheme (50 µl)
· 10 µl HF buffer
· 2 µl dNTPs Mix
· 4 µl Primer Mix (here rpsE)
· 1 µl RNA
· 0,5 µl PhuS
· 32 µl RNAse free H20.
PCR Programm
· 98°C 2 min
· 98°C 35 sec
· 52°C 35 sec
· 72°C 45 sec
· 72°C 10 min
· 15°C hold
· run a gel to check if everything went correctly
In this case we did this digestion with the wt, GP1344 and GP1346. Additionally we used a NTC and cDNA control.
Ø The digestion worked fine. No DNA detectable in our samples, only in the cDNA
Ø Determine RNA concentration
Ø This failed
We did the digestion again. This time we used 6 samples in total, two samples of each strain. IN the first three samples we used 5 µl DNAse I according to protocol, in the other three samples we only used 2.5 µl DNAse I.
Stored the samples over night at -20°C.
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Mini-Preps of RiboA Clones 1, 2, 4 and 5 (see gel from 12.08.), RD of Ribo 2,4,5, Ligations of the Riboswitches into next vectors, MiniPrep of RBSrev + pSB1C3, Prom1rev + part6.2, Prom3rev + part 6.2 (C1 – C3 each) and PCR test, PCR to test, if RBSrev C1 – C3 are positive or negative, Test restriction digest of Prom1rev + part6.2, Prom3rev + part 6.2 (C1 – C3 each)....
Cloning of Riboswitch A
Mini-Preps of RiboA Clones 1, 2, 4 and 5 (see gel from 12.08.) using the plasmid purification kit
Nanodrop measurements:
Sample | ng/µl | A260nm/A280nm | A260nm/A230nm |
Ribo A clone 1 | 213.9 | 1.89 | 1.86 |
Ribo A clone 2 | 152.5 | 1.95 | 2.00 |
Ribo A clone 4 | 396.6 | 1.90 | 2.11 |
Ribo A clone 5 | 429.4 | 1.89 | 2.18 |
Restriction Digestion of
Ribo A clones 4 +5, (R.D. with E + S)
Ribo B clone 2, (R.D. with X + P)
Ribo C clone 4, (R.D. with E + S)
Ribo D clone 5 (R.D. with X + P)
1500 ng DNA
3µl enzyme FD
3µl enzyme FD
4µl Buffer FD
40 µl reaction (filled up with H2O)
for 1.5 h at 37°C
Gel:
M/plasmid DNA Ribo A clone 4/ R.D. Ribo A clone 4/ plasmid DNA Ribo A clone 5/ R.D. Ribo A clone 5/ plasmid DNA Ribo B clone 2/ R.D. Ribo B clone 2/ plasmid DNA Ribo C clone 4/ R.D. Ribo C clone 4/ plasmid DNA Ribo D clone 5/ R.D. Ribo D clone 5
![](https://static.igem.org/mediawiki/2013/c/c12-2/Goe-19.08.13-RT-1.png)
→ loading of all R.D. Ribos A-D onto Gel for gel extraction
→ Gelextraxtion using the PCR purification kit, nanodrop measurements on the eppis (3-5ng/µl)
Ligations of the Riboswitches into next vectors
Ribo A clones 4 +5, (R.D. with E + S) with CFP and YFP (R.D. with E+X) (see 12.08.)
Ribo B clone 2, (R.D. with X + P) with Part1 and Part3 (R.D. with S+P) (see 12.08.)
Ribo C clone 4, (R.D. with E + S) with Part8 (R.D. with E+X) (see 12.08.)
Ribo D clone 5 (R.D. with X + P) with Part1 and Part3 (R.D. with S+P) (see 12.08.)
For that, all of the inserts (60ng) was used for ligation ino the Vectors (20ng)
0,75-1µl Vectors
12µl Insert (Ribo A-D)
2µl T4 Buffer
2µl T4 Ligase
3-3,25µl H20
20µl reaction
Incubation overnight at 16°C
MiniPrep of RBSrev + pSB1C3, Prom1rev + part6.2, Prom3rev + part 6.2 (C1 – C3 each)
- kit: Nucleospin, Macherey-Nagel
- 1st elution: 30 μl HPLC-water (pre-warmed), incubation for 2 min at 50 °C
- 2nd elution: 22 μl HPLC-water (pre-warmed), incubation for 2 min at 50 °C
- no concentration measurement, since NanoDrop is dirty and measures 6 – 7 ng DNA in HPLC water used as a blank before…
- samples stored in “DarR reporter system” - box
PCR to test, if RBSrev C1 – C3 are positive or negative
- RBSrev insert has only 12 bp → one would not see it in a gel
- in Colony PCR, there was for all clones a band at height of terminator band (re-ligand)
- perform PCR to test, if terminator band is really there/ if clones are positive
- PCR (similar to colony PCR, but with plasmids as a template):
Component | Volume |
Taq | 1 μl |
10x Taq buffer | 2.5 μl |
dNTP mix | 1 μl |
iGEM_38 (VF2) 1:20 | 1 μl |
iGEM_39 (VR) 1:20 | 1 μl |
dH2O | 17.5 μl |
template | 1 μl |
Total | 25 μl |
- of the plasmids prepped today (see MiniPrep), a 1:20 dilution was prepared, of this dilution, 1 μl was used a template (1 reaction for each clone)
- as an additional control, part 7 C1 plasmid (dilution from 15.6.13, 1:10) was used
- Neg. control: 1 μl water as a template
- preparation of a 6x MasterMix:
Component | Volum |
Taq | 6μl |
10x Taq buffe | 15μl |
dNTP mix | 6μl |
iGEM_38 (V2) 1:20 | 6μl |
iGEM_39 (VR) 1:20 | 6μl |
dH2O | 105μl |
Total | 144μl |
→ addition of 24 μl MasterMix to each sample
- PCR protocol: same as for colony-PCR
Test restriction digest of Prom1rev + part6.2, Prom3rev + part 6.2 (C1 – C3 each)
- of the plasmids prepped today, 2 μl were used for the digest, since the concentration could not be measured with NanoDrop
- one reaction contained
Component | Volume |
plasmid | 2μl |
10x FD Green buffer | 1μl |
EcoRI FD | 1μ |
PstI FD | 1μl |
dH2O | 5μl |
Total | 10μl |
- the plasmid was added to the tubes, then addition of 8 μl MasterMix; the MasterMix consisted of…
Component | Volume |
10x FD Green buffer | 7μl |
EcoRI FD | 7μl |
PstI FD | 7μl |
dH2O | 35μl |
Total | 56μl |
- incubation at 37 °C for 1 h
Gel run:
- 1 % agarose-1xTAE gel
- loading of 3 μl 2 log ladder
- loading of 1 μl of uncut plasmid + 3 μl dH2O + 1 μl 5xLD
- loading of 5 μl of RD reaction
- run at 100 V
- EtBr staining + destaining
- UV detection
Loading: Marker/Prom1rev + part6.2 C1 uncut/ Prom1rev + part6.2 C1 RD/ Prom1rev + part6.2 C2 uncut/ Prom1rev + part6.2 C2 RD/ Prom1rev + part6.2 C3 uncut/ Prom1rev + part6.2 C3 RD/ Prom3rev + part6.2 C1 uncut/ Prom3rev + part6.2 C1 RD/ Prom3rev + part6.2 C2 uncut/ Prom3rev + part6.2 C2 RD/ Prom3rev + part6.2 C3 uncut/ Prom3rev + part6.2 C3 RD/Marker
![](https://static.igem.org/mediawiki/2013/1/1d/Goe-19.08.13-RT-2.png)
part 6.2 Re-ligand: 35 bp (Promoter 3) + 8 bp (Scar between promoter and DarR operator) + 14 bp (operator) + 8 bp (Scar between operator and part 6 GFP) + 876 bp (part 6 GFP) = 941 → ca. 940 bp should be visible in addition to pSB1C3 (2070 bp)
part 6.2 C1 plasmid and Promoter1rev/Promoter3rev: 112 bp (rev. Promoter) + 8 bp (Scar between rev. promoter and promoter 3) + 35 bp (Promoter 3) + 8 bp (Scar between promoter and DarR operator) + 14 bp (operator) + 8 bp (Scar between operator and part 6 GFP) + 876 bp (part 6 GFP) = 1061 → ca. 1060 bp should be visible in addition to pSB1C3 (2070 bp)
Conclusion:
All clones seemed to be positive, since fragments have slightly more than 1 kb and ca. 2 kb. Sequencing of C1 and C2 of each.
Sequencing results from 16.8.13
Terminatorrev + pSB1C3 C1 and C3: both clones contain the correct sequence of the terminator, but in reverse direction
DarRrev + pSB1C3 C1:
- between NotI and PstI restriction site, an additional G is inserted
- 2nd base of 10th codon: A is missing
- → don’t work with this clone!
DarRrev + pSB1C3 C4:
- all mutations from the forward primer are present at correct site
- no other mutations observed
- → work with this clone
Restriction of Terminatorrev + pSB1C3 C1 for generating vector for ligation with DarRrev
15 μl (ca. 1500 ng) plasmid Termrev C1 (from 16.8.13, 102.8 ng/μl)
4 μl SpeI FD
4 μl FD buffer 10x
17 μl dH2O
in total 40 μl
- digest: 2 h at 37 °C
Restriction of DarRrev + pSB1C3 C4 for generating the insert for ligation with Termrev C1 vector
6 μl (ca. 1500 ng) plasmid Termrev C1 (from 16.8.13, 282.5 ng/μl)
3μl XbaI FD
3 μl PstI FD
4 μl FD buffer 10x
24μl dH2O
in total 40 μl
- digest: 2 h at 37 °C
Gel run for DarRrev + pSB1C3 C4 and Terminatorrev + pSB1C3 C1 RDs and RBSrev Test PCRs (all from today, see above)
- 1 % agarose-1xTAE gel
- loading of 3 μl 2 log ladder
- loading of 1 μl of uncut plasmid + 3 μl dH2O + 1 μl 5xLD
- loading of 5 μl of RD reaction
- loading of 5 μl PCR reaction (addition of 5 μl of 5xLD to each PCR sample)
- run at 100 V
- EtBr staining + destaining
- UV detection
Loading: Marker/Terminatorrev C1 uncut/Terminatorrev + SpeI RD/DarRrev C4 uncut/DarRrev C4 + XbaI and PstI/RBSrev PCR: neg. control/RBSrev PCR: part 7 C1 plasmid/RBSrev PCR: RBSrev C1/RBSrev PCR: RBSrev C2/RBSrev PCR: RBSrev C3/Marker
![](https://static.igem.org/mediawiki/2013/0/05/Goe-19.08.13-RT-3.png)
- Termrev C1 is digested incompletely, but since the DarRrev insert won’t be dephosphorylated, one can dephosphorylate the incompletely digested vector → purifiy vector and digest with PstI
- DarRrev C4 plasmid is digested completely → purify DarRrev fragment with gel extraction
- PCR: the plasmids part 7 C1 and the RBSrev C1, C2 and C3 showed the expected products, but the negative control had a band at ca. 200 – 300 bp (I’ve no explanation for that: the water I used was the same for all other reactions! And the other material as well. Could be Bacillus spores, unclean PCR tubes, or my DNA…? XD → forget negative control (some people even don’t do no-template controls…), since expected products dominate/bands on neg. control seem not to be in lanes of all other samples → prepare C1 for sequencing
Hybridization of RBSrev, Promoter1rev and Promoter3rev repeated
- RBSrev must be cloned into Prom1rev/Prom3rev + part6.2 (from now on termed part 6.3A/B)
- Promoter1rev and Promoter3rev must be cloned into shipping vector
- hybridization performed as on 13.08.2013
- samples stored at – 20 °C in DarR reporter system-box
Concentration measurement (NanoDrop worked again!) of plasmids purified today
![](https://static.igem.org/mediawiki/2013/3/3e/Goe-19.08.13-RT-4.png)
Preparation of samples for sequencing by SeqLab
- sample no. 3: Promoter1rev + part6.2 C1 (part 6.3 A C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)
- sample no. 4: Promoter1rev + part6.2 C2 (part 6.3 A C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)
- sample no. 5: Promoter3rev + part6.2 C1 (part 6.3 B C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)
- sample no. 6: Promoter3rev + part6.2 C2 (part 6.3 B C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)
- sample no. 7: RBSrev + pSB1C3 C1 + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)
PCR clean up of Termrev C1 + SpeI digest
- with Qiagen PCR purification kit
- 500 μl PB buffer used
- elution 1x with 30 μl HPLC water (pre-warmed), incubation at ca. 57°C for 2 min
sample stored at – 20 °C in DarR reporter system box, marked with green
Gel ex of DarRrev insert
- 1%-agarose-1xTAE gel
- addition of 9 μl 5x LD to 37 μl reaction
- loading of whole reaction on gel
- loading of 3 μl marker
- loading of 1 μl uncut DarRrev C4 plasmid + 3 μl dH2O + 1 μl 5xLD
- run at 85 V
- 5 min staining with EtBr, short destaining in water, UV detection briefly, then gel ex under low UV light, tried to do gel ex, but signal was too weak, so again brief staining and destaining, then gel ex was possible
Gel after gel ex:
![](https://static.igem.org/mediawiki/2013/e/e1/Goe-19.08.13-RT-5.png)
- a bit was lost, but that’s ok…
- gel pieces weighed and stored in DarR reporter system box at – 20 °C
Protein expression for crystallization of L. monocytogenes DAC domain
Protein expression for crystallization of L. monocytogenes DAC domain
- Preparation of 10l LB medium
- Inoculation of 2x 50ml LB medium containing ampicillin with 1013 cryo stock
- Incubation o/n at 37°C with agitation
- Measurement of OD600
- Inoculation of 10x 1l LB medium containing ampicillin with culture having OD600=0,1
- Incubation at 37°C until OD600=0,5-0,7
- Add each 1ml of 1M IPTG to each 1l of culture
- Incubation for 12-14h at 16°C with agitation
- Measurement of OD600 and determine protein concentration and save samples for SDS-PAGE
- Transfer each 500ml of cultures into centrifugation tubes and centrifuge for 10min at 5000rpm at 4°C
- Resuspend pellet in 20ml buffer W
- Pool solution of 2 pellets and transfer protein solution into falcon tube
- Centrifugation for 15min at 5000rpm at 4°C
- Discard supernatant and store pellet at -20°C
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Riboswitch A Clones 1, 2, 4 and 5 inocculated into 4ml Cm cultures for minipreps tomorrow
Protein expression for crystallization of L. monocytogenes DAC domain
Protein expression for crystallization of L. monocytogenes DAC domain
- Preparation of 10l LB medium
- Inoculation of 2x 50ml LB medium containing ampicillin with 1013 cryo stock
- Incubation o/n at 37°C with agitation
- Measurement of OD600
- Inoculation of 10x 1l LB medium containing ampicillin with culture having OD600=0,1
- Incubation at 37°C until OD600=0,5-0,7
- Add each 1ml of 1M IPTG to each 1l of culture
- Incubation for 12-14h at 16°C with agitation
- Measurement of OD600 and determine protein concentration and save samples for SDS-PAGE
- Transfer each 500ml of cultures into centrifugation tubes and centrifuge for 10min at 5000rpm at 4°C
- Resuspend pellet in 20ml buffer W
- Pool solution of 2 pellets and transfer protein solution into falcon tube
- Centrifugation for 15min at 5000rpm at 4°C
- Discard supernatant and store pellet at -20°C