Team:Goettingen/NoteBook w8

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<p class="c0"><span class="c5 c25">Miniprep of the Promoter-GFP Clones</span></p><p class="c0"><span class="c25">Minipreps performed with the Plasmid purification kit, elution in 30µl pre-warmed water, AW buffer step included</span></p><p class="c0"><span class="c25 c5">Nanodrop:</span></p><a href="#" name="95cf6dbdc2ddf167c2a70dd6379ba7b45ea80d19"></a><a href="#" name="2"></a><table cellpadding="0" cellspacing="0" class="c2"><tbody><tr><td class="c15"><p class="c4 c0"><span class="c9">Clone</span></p></td><td class="c13"><p class="c4 c0"><span class="c9 c5">Concentration (ng/µl)</span></p></td><td class="c17"><p class="c4 c0"><span class="c9 c5">A</span><span class="c8 c5">260nm</span><span class="c9 c5">/A</span><span class="c8 c5">280nm</span></p></td><td class="c1"><p class="c4 c0"><span class="c9 c5">A</span><span class="c5 c8">260nm</span><span class="c9 c5">/A</span><span class="c8 c5">230nm</span></p></td></tr><tr><td class="c15"><p class="c4 c0"><span class="c9">1</span></p></td><td class="c13"><p class="c4 c0"><span class="c9">233</span></p></td><td class="c17"><p class="c4 c0"><span class="c9">1,89</span></p></td><td class="c1"><p class="c4 c0"><span class="c9">2,2</span></p></td></tr><tr><td class="c15"><p class="c4 c0"><span class="c9">2</span></p></td><td class="c13"><p class="c4 c0"><span class="c9">208</span></p></td><td class="c17"><p class="c4 c0"><span class="c9">1,9</span></p></td><td class="c1"><p class="c4 c0"><span class="c9">2,2</span></p></td></tr><tr><td class="c15"><p class="c4 c0"><span class="c9">3</span></p></td><td class="c13"><p class="c4 c0"><span class="c9">191</span></p></td><td class="c17"><p class="c4 c0"><span class="c9">1,9</span></p></td><td class="c1"><p class="c4 c0"><span class="c9">2,1</span></p></td></tr><tr><td class="c15"><p class="c4 c0"><span class="c9">4</span></p></td><td class="c13"><p class="c4 c0"><span class="c9">209</span></p></td><td class="c17"><p class="c4 c0"><span class="c9">1,9</span></p></td><td class="c1"><p class="c4 c0"><span class="c9">2,0</span></p></td></tr><tr><td class="c15"><p class="c4 c0"><span class="c9">5</span></p></td><td class="c13"><p class="c0 c4"><span class="c9">207</span></p></td><td class="c17"><p class="c4 c0"><span class="c9">1,9</span></p></td><td class="c1"><p class="c4 c0"><span class="c9">2,2</span></p></td></tr><tr><td class="c15"><p class="c4 c0"><span class="c9">6</span></p></td><td class="c13"><p class="c4 c0"><span class="c9">179</span></p></td><td class="c17"><p class="c4 c0"><span class="c9">1,9</span></p></td><td class="c1"><p class="c4 c0"><span class="c9">2,2</span></p></td></tr><tr><td class="c15"><p class="c4 c0"><span class="c9">7</span></p></td><td class="c13"><p class="c4 c0"><span class="c9">241</span></p></td><td class="c17"><p class="c4 c0"><span class="c9">1,9</span></p></td><td class="c1"><p class="c4 c0"><span class="c9">1,7</span></p></td></tr><tr><td class="c15"><p class="c4 c0"><span class="c9">8</span></p></td><td class="c13"><p class="c4 c0"><span class="c9">141</span></p></td><td class="c17"><p class="c4 c0"><span class="c9">1,9</span></p></td><td class="c1"><p class="c4 c0"><span class="c9">2,2</span></p></td></tr></tbody></table><p class="c10 c0"><span class="c23 c5"></span></p><p class="c0"><span class="c5 c23">Plates with clones of Riboswitch, DarR and E0840 Clonation put into fridge for further processing on monday</span></p>

<p class="c0"><span class="c5">Promoter-GFP Clonation</span></p><p class="c0"><span class="c5">Photos</span><span>&nbsp;of the plates with Promoter-GFP in pSB1C3:</span></p><p class="c0"><span>GFP:</span></p><img src="https://static.igem.org/mediawiki/2013/f/f5/Goe-25.07-RT-1.png" width="500" /><p class="c0"><span>Through light:</span></p><img src="https://static.igem.org/mediawiki/2013/6/66/Goe-25.07-RT-2.png" width="500" /><p class="c0"><span>a lot of positive clones, but also negative ones.</span></p><p class="c0"><span>-&gt; 8 positive clones were picked and inoculated into 4ml Cam cultures for minipreps tomorrow</span></p><p class="c0"><span>-&gt; Since there was no religation control on any plates made by Bingyao, I could not check the vector without the Promoter for GFP. The green cultures could therefore also be religated vectors expressing GFP without the Promoter. Sequencing will show us what is in there.</span></p><p class="c0"><span>-&gt;</span><span class="c5">KEEP IN MIND:</span><span>&nbsp;</span><span class="c5 c11">ALLWAYS DO A RELIGATION CONTROL</span><span class="c11">&nbsp;</span><span class="c11 c5">AND TRANSFORM IT WITH THE OTHER LIGATIONS!</span></p><p class="c10 c0"><span class="c11 c5"></span></p>

<p class="c0"><span class="c5">Minipreps Part 1-4, Part 7</span></p><p class="c0"><span>eluted in HPLC water(30ul), measured by Nanodrop, (ng/ul)</span></p><a href="#" name="62a5ee52fa07f525aba6264d4c98e81fe2a034e1"></a><a href="#" name="0"></a><table cellpadding="0" cellspacing="0" class="c2"><tbody><tr><td class="c3"><p class="c14 c0"><span class="c5">&nbsp;epi</span></p></td><td class="c3"><p class="c0"><span class="c5">Part1</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">&nbsp;epi</span></p></td><td class="c3"><p class="c0"><span class="c5">Part2</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">&nbsp;epi</span></p></td><td class="c3"><p class="c0"><span class="c5">Part3</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">&nbsp;epi</span></p></td><td class="c3"><p class="c0"><span class="c5">Part4</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">&nbsp;epi</span></p></td><td class="c3"><p class="c0"><span class="c5">Part7</span></p></td></tr><tr><td class="c3"><p class="c0 c14"><span class="c5">1</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">241</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">1</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">114</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">1</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">252</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">1</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">122</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">1</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">121</span></p></td></tr><tr><td class="c3"><p class="c14 c0"><span class="c5">2</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">235</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">2</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">118</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">2</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">262</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">2</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">149</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">2</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">104</span></p></td></tr><tr><td class="c3"><p class="c14 c0"><span class="c5">3</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">211</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">3</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">100</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">3</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">278</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">3</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">151</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">3</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">132</span></p></td></tr><tr><td class="c3"><p class="c14 c0"><span class="c5">4</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">229</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">4</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">106</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">4</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">273</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">4</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">125</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">4</span></p></td><td class="c3"><p class="c14 c0"><span class="c5">92</span></p></td></tr></tbody></table><p class="c10 c0"><span class="c5"></span></p><p class="c0"><span class="c5">Gel of today´s PCR for the DarR biobrick:</span></p><p class="c0"><span class="c5">M|2µlPrimer|4µlPrimer</span></p><p class="c0"><span>Gel here:</span></p><img src="https://static.igem.org/mediawiki/2013/d/dc/Goe-24.07.13-RT.png" /><p class="c10 c0"><span></span></p><p class="c0"><span>-&gt;Cleanup of today´s PCR and yesterdays with the PCR purification kit, elution in 30µl prewarmed water</span></p><p class="c10 c0"><span></span></p><p class="c0"><span class="c5">Nanodrop:</span></p><a href="#" name="8425684373b9dc608d01e498189ae3be36add985"></a><a href="#" name="1"></a><table cellpadding="0" cellspacing="0" class="c2"><tbody><tr><td class="c16"><p class="c4 c0"><span>Reaction</span></p></td><td class="c12"><p class="c4 c0"><span class="c5">Concentration (ng/µl)</span></p></td><td class="c1"><p class="c4 c0"><span class="c5">A</span><span class="c5 c21">260nm</span><span class="c5">/A</span><span class="c21 c5">280nm</span></p></td><td class="c1"><p class="c4 c0"><span class="c5">A</span><span class="c21 c5">260nm</span><span class="c5">/A</span><span class="c21 c5">230nm</span></p></td></tr><tr><td class="c16"><p class="c4 c0"><span>2µl today</span></p></td><td class="c12"><p class="c4 c0"><span>11.8</span></p></td><td class="c1"><p class="c4 c0"><span>2.39</span></p></td><td class="c1"><p class="c4 c0"><span>1.65</span></p></td></tr><tr><td class="c16"><p class="c4 c0"><span>4µl today</span></p></td><td class="c12"><p class="c4 c0"><span>17</span></p></td><td class="c1"><p class="c4 c0"><span>2.04</span></p></td><td class="c1"><p class="c4 c0"><span>1.86</span></p></td></tr><tr><td class="c16"><p class="c4 c0"><span>1µl yesterday</span></p></td><td class="c12"><p class="c4 c0"><span>14.2</span></p></td><td class="c1"><p class="c4 c0"><span>2.02</span></p></td><td class="c1"><p class="c4 c0"><span>1.95</span></p></td></tr><tr><td class="c16"><p class="c4 c0"><span>2µl yesterday</span></p></td><td class="c12"><p class="c4 c0"><span>17</span></p></td><td class="c1"><p class="c4 c0"><span>2.28</span></p></td><td class="c1"><p class="c4 c0"><span>1.86</span></p></td></tr></tbody></table><p class="c0"><span class="c5">&nbsp;</span><span>-&gt;Stored in the “white sticker Ute” Box</span></p><p class="c10 c0"><span></span></p>

<p class="c0"><span class="c5">DarR PCR</span></p><p class="c0"><span class="c5">&nbsp;</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>DarR fusion PCR</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>After PCR cleanup, yield way to low. maybe mistake somewhere. Repeated on 24.7</span></p><p class="c0"><span class="c5">Ligation Part 6 + Part 3</span></p><p class="c0"><span class="c5">&nbsp;</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>Setup: 5 µl Part 6 (9,8 µl/ng), 10 µl Part 3 (23,5 µl/ng), 2 µl Buffer, 1 µl T4 Ligase, 2 µl H2O</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>Overnight ligation 16°C</span></p><p class="c0 c20"><span class="c5">→</span><span class="c5">transformation done by Bingyao next day</span></p><p class="c20 c0"><span>&nbsp;</span></p><p class="c0"><span class="c5">Ligation of Part 7 + 40/41 Riboswitch and Part 7 + DarR</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>Due to low concentrations of Backbone and insert, ligation was set up in 20 µl and with only 20 ng of BB.</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>both ligations with 2 setups. 5 µl and 10 µl of insert</span></p><p class="c0 c7"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>re-ligation done</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>overnight ligation 16°C</span></p><p class="c20 c0"><span class="c5">→</span><span class="c5">transformation done by Bingyao next day</span></p><p class="c0"><span class="c5">Inocculation of Cryostocks</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>Parts 1-4 and Part 7 inocculated in 4x4ml LB + Antibiotics overnight </span></p><p class="c10 c0"><span></span></p><p class="c0"><span class="c5">Continue the Restriction yesterday-Part 8 with PstI</span></p><p class="c0"><span>Add 2 additional SpeI into the system and digest another 1 hour, most DNA is cut. &lt;--continue the uncomplete first round digestion(SpeI)</span></p><p class="c0"><span>The second round of part8 digestion with fast PstI:</span></p><p class="c0"><span>Add 4ul PstI into the system</span></p><p class="c0"><span>Digest 2hours.</span></p><p class="c0"><span>Dephosphorylation</span></p><p class="c0"><span>&nbsp; &nbsp; Add 2ul of fast AP into the system(fast AP is compatible with FD buffer)</span></p><p class="c0"><span>&nbsp; &nbsp; Reaction on 37 degree, 30min</span></p><p class="c0"><span>Purification with PCR clean up kit</span></p><p class="c0"><span>&nbsp; &nbsp; Final concentration : on the tube, about 28ng/ul;</span></p><p class="c10 c0"><span></span></p>

<p class="c0"><span class="c5">Innoculate ON culture for plasmid mini-prep</span></p><p class="c19 c0"><span>Part7 C1</span></p><p class="c19 c0"><span>RBS-GFP-Term construct C1,C4,C5</span></p><p class="c0"><span class="c5">Test restriction of plasmid RBS-GFP-Term:</span></p><p class="c0 c19"><span>C2 as control, C1,C4,C5</span></p><p class="c0"><span class="c26 c5 c27">&nbsp;---&gt;the Part6 already has the RBS and Term.</span></p><p class="c0"><span class="c26">&nbsp;</span><span>[that simply makes the work above meaningless]</span></p><p class="c0"><span class="c22 c5">For the construct of &nbsp;RBS-DarR ORF-Term construct (expression construct)</span></p><p class="c0 c18"><span class="c22 c5">DarR ORF into Part8(PBS plasmid)</span></p><p class="c0 c18"><span class="c5 c22">&nbsp; &nbsp; &nbsp;suffix insertion { cut part8 with SpeI and PstI, and the DarR ORF with XbalI and PstI }</span></p><p class="c0 c18"><span class="c22 c5">RBS-DarR ORF into part7(Term plasmid)</span></p><p class="c0 c18"><span class="c22 c5">&nbsp; &nbsp; &nbsp;Prefix insertion {cut part7 with EcoRI and XbaI, the RBS-DarR ORF with EcoRI and SpeI)</span></p><p class="c0 c18"><span class="c22 c5">&nbsp;</span></p><p class="c0"><span class="c5">The first round of part8 digestion with SpeI: to have linearized structure for </span><span class="c5">DarR ORF into Part8</span></p><p class="c0"><span>&nbsp; &nbsp; &nbsp; &nbsp; Fast SpeI: 4ul</span></p><p class="c0"><span>&nbsp; &nbsp; &nbsp; &nbsp; FD buffer : 4ul</span></p><p class="c0"><span>&nbsp; &nbsp; &nbsp; &nbsp; part8 DNA: 1.5ug</span></p><p class="c0"><span>&nbsp; &nbsp; &nbsp; &nbsp; Water: fill up to 40ul</span></p><p class="c19 c0"><span>Digestion:1.5 hour, not fully digested</span></p><p class="c0"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Stored at -20 overnight</span></p><p class="c0"><span>&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;tbc</span></p><p class="c10 c0"><span></span></p><p class="c0"><span class="c5">Restriction digestion of part 7</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>template from stock</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>restriction with EcoRI and PstI FD, gel extraction to remove terminator out of the construct</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>Nanodrop meassurment 5,3 ng/µl</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>Inocculation from cryostock. 5h, only 2h on the shaker (stupid me). Yield after miniprep way to low (10x 50 µl with 1-3 ng/µl)</span></p><p class="c7 c0"><span>-</span><span class="c6">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>→</span><span>redone on 23.7 overnight inocculation</span></p><p class="c0"><span>(pic to be added)</span></p><p class="c0 c10"><span></span></p>