Team:Goettingen/NoteBook
From 2013.igem.org
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<div class="monat">June</div> | <div class="monat">June</div> | ||
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+ | <div class="tlob" id="tl_0706"> | ||
+ | <span class="date"><img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <p class="timeline-title">Plasmid mini-prep for Part1-7</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <b>Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):</b> | ||
+ | <ul> | ||
+ | <li>900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)</li> | ||
+ | <li>vortex</li> | ||
+ | <li>store at – 70 °C (red box)</li> | ||
+ | </ul> | ||
+ | <b>Plasmid Mini-Preparation of parts 1 - 7:</b> | ||
+ | <p>ON cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol for purification of high copy plasmids</p> | ||
+ | <p>Step 5: recommended washing of silica membrane with buffer AW was performed</p> | ||
+ | <p>Step 7: Elution with buffer AE pre-heated to 50 – 60 °C (in future: DON’T elute with this buffer, use pre-heated HPLC-H2O instead! No one knows what’s inside the buffer and its components (EDTA?) could interfere with sequencing and other reactions)</p> | ||
+ | <br / > | ||
+ | <p>NanoDrop – Plasmid concentrations</p> | ||
+ | <table cellspacing="0"> | ||
+ | <tr><th>Part Number</th><th>c(DNA)[ng/μl]</th><th>A<sub>260</sub>/A<sub>280</sub></th><th>A<sub>260</sub>/A<sub>230</sub></th> | ||
+ | <tr><td>1</td><td>84.6</td><td>1.94</td><td>2.18</td></tr> | ||
+ | <tr><td>2</td><td>79.8</td><td>1.94</td><td>2.10</td></tr> | ||
+ | <tr><td>3</td><td>151.0</td><td>1.89</td><td>2.24</td></tr> | ||
+ | <tr><td>4</td><td>29.5</td><td>1.91</td><td>2.07</td></tr> | ||
+ | <tr><td>5</td><td>154.9</td><td>1.88</td><td>2.25</td></tr> | ||
+ | <tr><td>6</td><td>88.9</td><td>1.92</td><td>2.08</td></tr> | ||
+ | <tr><td>7</td><td>5.9</td><td>14.08</td><td>1.87</td></tr> | ||
+ | </table> | ||
+ | <p>Stored in red box at - 20°C</p> | ||
+ | <b>Primers arrived!</b> | ||
+ | <p>iGEM_32 ~ 37: dissolved in HPLC water, stored at -20oC in our box</p> | ||
+ | <p>100uM stock , for PCR dilute 1:20 in HPLC water.</p> | ||
+ | <p>Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
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+ | <div class="tlob" id="tl_0606"> | ||
+ | <span class="date">06th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <p class="timeline-title">Pick the colonies of part1-7</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <b>Media preparation</b> | ||
+ | <p>1000ml LB+Amp Solid medium => about 50 Plates(with black code)</p> | ||
+ | <b>Transformation</b> | ||
+ | <table cellspacing="0"> | ||
+ | <tr><th>Entry Numer</th><th>Location on the kit</th></tr> | ||
+ | <tr><td>BBa_B0034</td><td>P5 2 M</td></tr> | ||
+ | </table> | ||
+ | <b>Pick the colonies of Part1-7</b> | ||
+ | <p>4ml LB with antibiotics, overnight culture for mini-prep[marked with C1]</p> | ||
+ | <p>Backup plates:marked with C1,C2,C3 for each part.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
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Revision as of 07:10, 18 August 2013