Team:Goettingen/NoteBook w12
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+ | <p class="timeline-title">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)....</p> | ||
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+ | <p class="c14 c24"><span class="c2">Cloning of Riboswitch A </span></p><p class="c14 c24"><span class="c2">Mini-Preps of RiboA Clones 1, 2, 4 and 5 (see gel from 12.08.) using the plasmid purification kit</span></p><p class="c14 c24"><span>Nanodrop measurements:</span></p><a href="#" name="1125b986674db083e9c9e43e752e86645c46af29"></a><a href="#" name="0"></a><table cellpadding="0" cellspacing="0" class="c27"><tbody><tr><td class="c54"><p class="c4"><span class="c2">Sample</span></p></td><td class="c52"><p class="c4"><span class="c2">ng/µl</span></p></td><td class="c38"><p class="c4"><span class="c2">A</span><span class="c1">260nm</span><span class="c2">/A</span><span class="c1">280nm</span></p></td><td class="c41"><p class="c4"><span class="c2">A</span><span class="c1">260nm</span><span class="c2">/A</span><span class="c1">230nm</span></p></td></tr><tr><td class="c54"><p class="c4"><span>Ribo A clone 1</span></p></td><td class="c52"><p class="c4"><span>213.9</span></p></td><td class="c38"><p class="c4"><span>1.89</span></p></td><td class="c41"><p class="c4"><span>1.86</span></p></td></tr><tr><td class="c54"><p class="c4"><span>Ribo A clone 2</span></p></td><td class="c52"><p class="c4"><span>152.5</span></p></td><td class="c38"><p class="c4"><span>1.95</span></p></td><td class="c41"><p class="c4"><span>2.00</span></p></td></tr><tr><td class="c54"><p class="c4"><span>Ribo A clone 4</span></p></td><td class="c52"><p class="c4"><span>396.6</span></p></td><td class="c38"><p class="c4"><span>1.90</span></p></td><td class="c41"><p class="c4"><span>2.11</span></p></td></tr><tr><td class="c54"><p class="c4"><span>Ribo A clone 5</span></p></td><td class="c52"><p class="c4"><span>429.4</span></p></td><td class="c38"><p class="c4"><span>1.89</span></p></td><td class="c41"><p class="c4"><span>2.18</span></p></td></tr></tbody></table><p class="c14 c24"><span class="c2"> </span></p><p class="c14 c24"><span class="c2">Restriction Digestion of</span></p><p class="c14 c24"><span class="c2">Ribo A clones 4 +5, </span><span>(R.D. with E + S)</span></p><p class="c14 c24"><span class="c2">Ribo B clone 2, </span><span>(R.D. with X + P)</span></p><p class="c14 c24"><span class="c2">Ribo C clone 4, </span><span>(R.D. with E + S)</span></p><p class="c14 c24"><span class="c2">Ribo D clone 5</span><span> (R.D. with X + P)</span></p><p class="c14 c24"><span class="c2"> </span></p><p class="c14 c24"><span>1500 ng DNA</span></p><p class="c14 c24"><span>3µl enzyme FD</span></p><p class="c14 c24"><span>3µl enzyme FD</span></p><p class="c14 c24"><span>4µl Buffer FD</span></p><p class="c14 c24"><span>40 µl reaction (filled up with H</span><span class="c10">2</span><span>O)</span></p><p class="c14 c24"><span>for 1.5 h at 37°C</span></p><p class="c14 c24"><span> </span></p><p class="c14 c24"><span class="c2">Gel:</span></p><p class="c14 c24"><span class="c2">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/c2/Goe-19.08.13-RT-1.png" /><p class="c8 c24"><span class="c2"></span></p><p class="c14 c24"><span>→ loading of all R.D. Ribos A-D onto Gel for gel extraction </span></p><p class="c14 c24"><span>→ Gelextraxtion using the PCR purification kit, nanodrop measurements on the eppis (3-5ng/µl)</span></p><p class="c8 c24"><span></span></p><p class="c14 c24"><span class="c2">Ligations of the Riboswitches into next vectors </span></p><p class="c14 c24"><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="c14 c24"><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="c14 c24"><span>Ribo C clone 4, (R.D. with E + S) with Part8 (R.D. with E+X) (see 12.08.)</span></p><p class="c14 c24"><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="c14 c24"><span>For that, all of the inserts (60ng) was used for ligation ino the Vectors (20ng) </span></p><p class="c14 c43"><span>0,75-1µl Vectors</span></p><p class="c14 c43"><span>12µl Insert (Ribo A-D)</span></p><p class="c14 c43"><span>2µl T4 Buffer</span></p><p class="c14 c43"><span>2µl T4 Ligase</span></p><p class="c14 c43"><span>3-3,25µl H20</span></p><p class="c14 c43"><span>20µl reaction </span></p><p class="c14 c24"><span>Incubation overnight at 16°C</span></p><p class="c8 c24"><span></span></p><p class="c14 c24"><span class="c2">MiniPrep of RBS</span><span class="c1">rev</span><span class="c2"> + pSB1C3, Prom1</span><span class="c1">rev</span><span class="c2"> + part6.2, Prom3</span><span class="c1">rev</span><span class="c2"> + part 6.2 (C1 – C3 each) </span></p><p class="c9"><span>-</span><span> </span><span>kit: Nucleospin, Macherey-Nagel</span></p><p class="c9"><span>-</span><span> </span><span>1</span><span class="c5">st</span><span> elution: 30 μl HPLC-water (pre-warmed), incubation for 2 min at 50 °C</span></p><p class="c9"><span>-</span><span> </span><span>2</span><span class="c5">nd</span><span> elution: 22 μl HPLC-water (pre-warmed), incubation for 2 min at 50 °C</span></p><p class="c9"><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="c9"><span>-</span><span> </span><span>samples stored in “DarR reporter system” - box</span></p><p class="c3"><span></span></p><p class="c8"><span></span></p><p class="c14"><span class="c2">PCR to test, if RBS</span><span class="c1">rev</span><span class="c2"> C1 – C3 are positive or negative</span><span> </span></p><p class="c9"><span>-</span><span> </span><span>RBS</span><span class="c10">rev</span><span> insert has only 12 bp → one would not see it in a gel</span></p><p class="c9"><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="c9"><span>-</span><span> </span><span>perform PCR to test, if terminator band is really there/ if clones are positive</span></p><p class="c9"><span>-</span><span> </span><span>PCR (similar to colony PCR, but with plasmids as a template):</span></p><p class="c3"><span></span></p><a href="#" name="7fe2bd2d13d171a3c9284fc10b2cae22ea6ce0c3"></a><a href="#" name="1"></a><table cellpadding="0" cellspacing="0" class="c27"><tbody><tr><td class="c51"><p class="c16 c14"><span class="c2">Component</span></p></td><td class="c12"><p class="c7 c16"><span class="c2">Volume</span></p></td></tr><tr><td class="c51"><p class="c16 c14"><span class="c2">Taq</span></p></td><td class="c12"><p class="c7 c16"><span>1 μl</span></p></td></tr><tr><td class="c51"><p class="c16 c14"><span class="c2">10x Taq buffer</span></p></td><td class="c12"><p class="c7 c16"><span>2.5 μl</span></p></td></tr><tr><td class="c51"><p class="c16 c14"><span class="c2">dNTP mix</span></p></td><td class="c12"><p class="c7 c16"><span>1 μl</span></p></td></tr><tr><td class="c51"><p class="c16 c14"><span class="c2">iGEM_38 (VF2) 1:20</span></p></td><td class="c12"><p class="c7 c16"><span>1 μl</span></p></td></tr><tr><td class="c51"><p class="c14 c16"><span class="c2">iGEM_39 (VR) 1:20</span></p></td><td class="c12"><p class="c7 c16"><span>1 μl</span></p></td></tr><tr><td class="c51"><p class="c16 c14"><span class="c2">dH</span><span class="c1">2</span><span class="c2">O</span></p></td><td class="c12"><p class="c7 c16"><span>17.5 μl</span></p></td></tr><tr><td class="c51"><p class="c16 c14"><span class="c2">template</span></p></td><td class="c12"><p class="c7 c16"><span>1 μl</span></p></td></tr><tr><td class="c51"><p class="c16 c14"><span class="c2">Total</span></p></td><td class="c12"><p class="c7 c16"><span>25 μl</span></p></td></tr></tbody></table><p class="c14"><span class="c2"> </span></p><p class="c14"><span class="c2"> </span></p><p class="c9"><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="c9"><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="c9"><span>-</span><span> </span><span>Neg. control: 1 μl water as a template</span></p><p class="c9"><span>-</span><span> </span><span>preparation of a 6x MasterMix:</span></p><p class="c3"><span></span></p><p class="c14"><span> </span></p><a href="#" name="e36506b49dad64a763ef3905bc5a07c7901d0766"></a><a href="#" name="2"></a><table cellpadding="0" cellspacing="0" class="c27"><tbody><tr><td class="c21"><p class="c18 c14"><span class="c2">Component</span></p></td><td class="c20"><p class="c18 c14"><span class="c2">Volum</span></p></td></tr><tr><td class="c21"><p class="c18 c14"><span class="c2">Taq</span></p></td><td class="c20"><p class="c0"><span>6μl</span></p></td></tr><tr><td class="c21"><p class="c18 c14"><span class="c2">10x Taq buffe</span></p></td><td class="c20"><p class="c0"><span>15μl</span></p></td></tr><tr><td class="c21"><p class="c18 c14"><span class="c2">dNTP mix</span></p></td><td class="c20"><p class="c0"><span>6μl</span></p></td></tr><tr><td class="c21"><p class="c18 c14"><span class="c2">iGEM_38 (V2) 1:20</span></p></td><td class="c20"><p class="c0"><span>6μl</span></p></td></tr><tr><td class="c21"><p class="c18 c14"><span class="c2">iGEM_39 (VR) 1:20</span></p></td><td class="c20"><p class="c0"><span>6μl</span></p></td></tr><tr><td class="c21"><p class="c18 c14"><span class="c2">dH</span><span class="c1">2</span><span class="c2">O</span></p></td><td class="c20"><p class="c0"><span>105μl</span></p></td></tr><tr><td class="c21"><p class="c14 c18"><span class="c2">Total</span></p></td><td class="c20"><p class="c0"><span>144μl</span></p></td></tr></tbody></table><p class="c15 c14"><span>→ addition of 24 μl MasterMix to each sample </span></p><p class="c9"><span>-</span><span> </span><span>PCR protocol: same as for colony-PCR</span></p><p class="c3"><span></span></p><p class="c3"><span></span></p><p class="c14"><span class="c2">Test restriction digest of Prom1</span><span class="c1">rev</span><span class="c2"> + part6.2, Prom3</span><span class="c1">rev</span><span class="c2"> + part 6.2 (C1 – C3 each)</span><span> </span></p><p class="c9"><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="c9"><span>-</span><span> </span><span>one reaction contained</span></p><p class="c3"><span></span></p><a href="#" name="30c7c869090768ae28bf86bdb7425dd700812cc5"></a><a href="#" name="3"></a><table cellpadding="0" cellspacing="0" class="c27"><tbody><tr><td class="c34"><p class="c7"><span class="c2">Component</span></p></td><td class="c42"><p class="c7"><span class="c2">Volume</span></p></td></tr><tr><td class="c34"><p class="c7"><span class="c2">plasmid</span></p></td><td class="c42"><p class="c7"><span>2μl</span></p></td></tr><tr><td class="c34"><p class="c7"><span class="c2">10x FD Green buffer</span></p></td><td class="c42"><p class="c7"><span>1μl</span></p></td></tr><tr><td class="c34"><p class="c7"><span class="c2">EcoRI FD</span></p></td><td class="c42"><p class="c7"><span>1μ</span></p></td></tr><tr><td class="c34"><p class="c7"><span class="c2">PstI FD</span></p></td><td class="c42"><p class="c7"><span>1μl</span></p></td></tr><tr><td class="c34"><p class="c7"><span class="c2">dH</span><span class="c1">2</span><span class="c2">O</span></p></td><td class="c42"><p class="c7"><span>5μl</span></p></td></tr><tr><td class="c34"><p class="c7"><span class="c2">Total</span></p></td><td class="c42"><p class="c7"><span>10μl</span></p></td></tr></tbody></table><p class="c7"><span> </span></p><p class="c9"><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="c3"><span></span></p><a href="#" name="29b8402d804b319bd43369e3e12060c61cd63977"></a><a href="#" name="4"></a><table cellpadding="0" cellspacing="0" class="c27"><tbody><tr class="c61"><td class="c6"><p class="c7"><span class="c2">Component</span></p></td><td class="c45"><p class="c7"><span class="c2">Volume</span></p></td></tr><tr><td class="c6"><p class="c7"><span class="c2">10x FD Green buffer</span></p></td><td class="c45"><p class="c7"><span>7μl</span></p></td></tr><tr><td class="c6"><p class="c7"><span class="c2">EcoRI FD</span></p></td><td class="c45"><p class="c7"><span>7μl</span></p></td></tr><tr><td class="c6"><p class="c7"><span class="c2">PstI FD</span></p></td><td class="c45"><p class="c7"><span>7μl</span></p></td></tr><tr><td class="c6"><p class="c7"><span class="c2">dH</span><span class="c1">2</span><span class="c2">O</span></p></td><td class="c45"><p class="c7"><span>35μl</span></p></td></tr><tr><td class="c6"><p class="c7"><span class="c2">Total</span></p></td><td class="c45"><p class="c7"><span>56μl</span></p></td></tr></tbody></table><p class="c14"><span> </span></p><p class="c14"><span> </span></p><p class="c9"><span>-</span><span> </span><span>incubation at 37 °C for 1 h</span></p><p class="c14 c43"><span> </span></p><p class="c14 c24"><span class="c2">Gel run:</span></p><p class="c14 c43"><span> </span></p><p class="c9"><span>-</span><span> </span><span>1 % agarose-1xTAE gel</span></p><p class="c9"><span>-</span><span> </span><span>loading of 3 μl 2 log ladder</span></p><p class="c9"><span>-</span><span> </span><span>loading of 1 μl of uncut plasmid + 3 μl dH</span><span class="c10">2</span><span>O + 1 μl 5xLD</span></p><p class="c9"><span>-</span><span> </span><span>loading of 5 μl of RD reaction</span></p><p class="c9"><span>-</span><span> </span><span>run at 100 V</span></p><p class="c9"><span>-</span><span> </span><span>EtBr staining + destaining</span></p><p class="c9"><span>-</span><span> </span><span>UV detection</span></p><p class="c14"><span> </span><span class="c2">Loading:</span><span> Marker/Prom1</span><span class="c10">rev</span><span> + part6.2 C1 uncut/ Prom1</span><span class="c10">rev</span><span> + part6.2 C1 RD/ Prom1</span><span class="c10">rev</span><span> + part6.2 C2 uncut/ Prom1</span><span class="c10">rev</span><span> + part6.2 C2 RD/ Prom1</span><span class="c10">rev</span><span> + part6.2 C3 uncut/ Prom1</span><span class="c10">rev</span><span> + part6.2 C3 RD/ Prom3</span><span class="c10">rev</span><span> + part6.2 C1 uncut/ Prom3</span><span class="c10">rev</span><span> + part6.2 C1 RD/ Prom3</span><span class="c10">rev</span><span> + part6.2 C2 uncut/ Prom3</span><span class="c10">rev</span><span> + part6.2 C2 RD/ Prom3</span><span class="c10">rev</span><span> + part6.2 C3 uncut/ Prom3</span><span class="c10">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="c14"><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="c2">ca. 940 bp should be visible in addition to pSB1C3 (2070 bp)</span></p><p class="c14"><span>part 6.2 C1 plasmid and Promoter1</span><span class="c10">rev</span><span>/Promoter3</span><span class="c10">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="c2">ca. 1060 bp should be visible in addition to pSB1C3 (2070 bp)</span></p><p class="c14"><span class="c2"> </span></p><p class="c14"><span class="c2 c55">Conclusion: </span></p><p class="c14"><span class="c55 c2">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="c8"><span class="c55 c2"></span></p><p class="c14"><span class="c2">Sequencing results from 16.8.13</span></p><p class="c14 c43"><span class="c2">Terminator</span><span class="c1">rev</span><span class="c2"> + pSB1C3 C1 and C3: </span><span>both clones contain the correct sequence of the terminator, but in reverse direction</span></p><p class="c9"><span class="c2">DarR</span><span class="c1">rev</span><span class="c2"> + pSB1C3 C1:</span></p><p class="c9"><span>-</span><span> </span><span>between NotI and PstI restriction site, an additional G is inserted</span></p><p class="c9"><span>-</span><span> </span><span>2</span><span class="c5">nd</span><span> base of 10</span><span class="c5">th</span><span> codon: A is missing</span></p><p class="c9"><span>-</span><span> → </span><span>don’t work with this clone!</span><span class="c2"> </span></p><p class="c9"><span class="c2">DarR</span><span class="c1">rev</span><span class="c2"> + pSB1C3 C4:</span></p><p class="c9"><span>-</span><span> </span><span>all mutations from the forward primer are present at correct site</span></p><p class="c9"><span>-</span><span> </span><span>no other mutations observed</span></p><p class="c9"><span>-</span><span> → </span><span> work with this clone</span></p><p class="c3"><span class="c2"></span></p><p class="c8"><span></span></p><p class="c14"><span class="c2">Restriction of Terminator</span><span class="c1">rev</span><span class="c2"> + pSB1C3 C1 for generating vector for ligation with DarR</span><span class="c1">re</span><span class="c10">v</span><span> </span></p><p class="c14 c43"><span>15 μl (ca. 1500 ng) plasmid Term</span><span class="c10">rev</span><span> C1 (from 16.8.13, 102.8 ng/μl)</span></p><p class="c14 c43"><span>4 μl SpeI FD</span></p><p class="c14 c43"><span>4 μl FD buffer 10x</span></p><p class="c14 c43"><span>17 μl dH</span><span class="c10">2</span><span>O</span></p><p class="c14 c43"><span>in total 40 μl</span></p><p class="c9"><span>-</span><span> </span><span>digest: 2 h at 37 °C</span></p><p class="c3"><span></span></p><p class="c14 c24"><span class="c2">Restriction of DarR</span><span class="c1">rev</span><span class="c2"> + pSB1C3 C4 for generating the insert for ligation with Term</span><span class="c1">rev</span><span class="c2"> C1 vector </span></p><p class="c14 c43"><span>6 μl (ca. 1500 ng) plasmid Term</span><span class="c10">rev</span><span> C1 (from 16.8.13, 282.5 ng/μl)</span></p><p class="c14 c43"><span>3μl XbaI FD</span></p><p class="c14 c43"><span>3 μl PstI FD</span></p><p class="c14 c43"><span>4 μl FD buffer 10x</span></p><p class="c14 c43"><span>24μl dH</span><span class="c10">2</span><span>O</span></p><p class="c14 c43"><span>in total 40 μl </span></p><p class="c9"><span>- </span><span>digest: 2 h at 37 °C</span></p><p class="c8"><span></span></p><p class="c14"><span class="c2">Gel run for DarR</span><span class="c1">rev</span><span class="c2"> + pSB1C3 C4 and Terminator</span><span class="c1">rev</span><span class="c2"> + pSB1C3 C1 RDs and RBS</span><span class="c1">rev</span><span class="c2"> Test PCRs (all from today, see above)</span><span> </span></p><p class="c9"><span>-</span><span> </span><span>1 % agarose-1xTAE gel</span></p><p class="c9"><span>-</span><span> </span><span>loading of 3 μl 2 log ladder</span></p><p class="c9"><span>-</span><span> </span><span>loading of 1 μl of uncut plasmid + 3 μl dH</span><span class="c10">2</span><span>O + 1 μl 5xLD</span></p><p class="c9"><span>-</span><span> </span><span>loading of 5 μl of RD reaction</span></p><p class="c9"><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="c9"><span>-</span><span> </span><span>run at 100 V</span></p><p class="c9"><span>-</span><span> </span><span>EtBr staining + destaining</span></p><p class="c9"><span>-</span><span> </span><span>UV detection</span></p><p class="c14"><span class="c2">Loading: </span><span>Marker/Terminator</span><span class="c10">rev</span><span> C1 uncut/Terminator</span><span class="c10">rev</span><span> + SpeI RD/DarR</span><span class="c10">rev</span><span> C4 uncut/DarR</span><span class="c10">rev</span><span> C4 + XbaI and PstI/RBS</span><span class="c10">rev</span><span> PCR: neg. control/RBS</span><span class="c10">rev</span><span> PCR: part 7 C1 plasmid/RBS</span><span class="c10">rev</span><span> PCR: RBS</span><span class="c10">rev</span><span> C1/RBS</span><span class="c10">rev</span><span> PCR: RBS</span><span class="c10">rev</span><span> C2/RBS</span><span class="c10">rev</span><span> PCR: RBS</span><span class="c10">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="c9"><span>-</span><span> </span><span>Term</span><span class="c10">rev</span><span> C1 is digested incompletely, but since the DarR</span><span class="c10">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="c9"><span>-</span><span> </span><span>DarR</span><span class="c10">rev</span><span> C4 plasmid is digested completely → purify DarR</span><span class="c10">rev</span><span> fragment with gel extraction</span></p><p class="c9"><span>-</span><span> </span><span>PCR: the plasmids part 7 C1 and the RBS</span><span class="c10">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="c3"><span></span></p><p class="c3"><span></span></p><p class="c14"><span class="c2">Hybridization of RBS</span><span class="c1">rev</span><span class="c2">, Promoter1</span><span class="c1">rev</span><span class="c2"> and Promoter3</span><span class="c1">rev</span><span class="c2"> repeated </span></p><p class="c9"><span>-</span><span> </span><span>RBS</span><span class="c10">rev</span><span> must be cloned into Prom1</span><span class="c10">rev</span><span>/Prom3</span><span class="c10">rev</span><span> + part6.2 (from now on termed part 6.3A/B)</span></p><p class="c9"><span>-</span><span> </span><span>Promoter1</span><span class="c10">rev</span><span> and Promoter3</span><span class="c10">rev</span><span> must be cloned into shipping vector</span></p><p class="c9"><span>-</span><span> </span><span>hybridization performed as on 13.08.2013</span></p><p class="c9"><span>-</span><span> </span><span>samples stored at – 20 °C in DarR reporter system-box</span></p><p class="c8"><span></span></p><p class="c14"><span class="c2">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="c8"><span></span></p><p class="c14"><span class="c2">Preparation of samples for sequencing by SeqLab</span></p><p class="c9"><span>-</span><span> </span><span>sample no. 3: Promoter1</span><span class="c10">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="c9"><span>-</span><span> </span><span>sample no. 4: Promoter1</span><span class="c10">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="c9"><span>-</span><span> </span><span>sample no. 5: Promoter3</span><span class="c10">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="c9"><span>-</span><span> </span><span>sample no. 6: Promoter3</span><span class="c10">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="c9"><span>-</span><span> </span><span>sample no. 7: RBS</span><span class="c10">rev</span><span> + pSB1C3 C1 + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)</span></p><p class="c3"><span class="c2"></span></p><p class="c8"><span></span></p><p class="c14"><span class="c2">PCR clean up of Term</span><span class="c1">rev</span><span class="c2"> C1 + SpeI digest </span></p><p class="c9"><span>-</span><span> </span><span>with Qiagen PCR purification kit</span></p><p class="c9"><span>-</span><span> </span><span>500 μl PB buffer used</span></p><p class="c9"><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="c9"><span>sample stored at – 20 °C in DarR reporter system box, marked with green </span></p><p class="c14 c24"><span class="c2">Gel ex of DarR</span><span class="c1">rev</span><span class="c2"> insert</span><span> </span></p><p class="c9"><span>-</span><span> </span><span>1%-agarose-1xTAE gel</span></p><p class="c9"><span>-</span><span> </span><span>addition of 9 μl 5x LD to 37 μl reaction</span></p><p class="c9"><span>-</span><span> </span><span>loading of whole reaction on gel</span></p><p class="c9"><span>-</span><span> </span><span>loading of 3 μl marker</span></p><p class="c9"><span>-</span><span> </span><span>loading of 1 μl uncut DarR</span><span class="c10">rev</span><span> C4 plasmid + 3 μl dH</span><span class="c10">2</span><span>O + 1 μl 5xLD</span></p><p class="c9"><span>-</span><span> </span><span>run at 85 V</span></p><p class="c9"><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="c14"><span class="c2">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="c9"><span>-</span><span> </span><span>a bit was lost, but that’s ok…</span></p><p class="c9"><span>-</span><span> </span><span>gel pieces weighed and stored in DarR reporter system box at – 20 °C</span></p><p class="c3"><span></span></p><p class="c8"><span></span></p> | ||
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Revision as of 10:28, 23 September 2013
![](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/c2/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
![](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