Team:Goettingen/week12-3
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<h2><b>V07_16 </b></h2><br> | <h2><b>V07_16 </b></h2><br> | ||
- | <b> | + | <b>Starpoint of the 2<sup>nd</sup> round of saturated mutagenesis PCR!</b><br> |
<ul> | <ul> | ||
- | <li>Experiment: <br> | + | <li>Experiment: <br>For the second round of saturated mutagenesis PCR we used the primers Tar6973Fw and Tar6973Rv. Here, aa residues 69 and 73 will be mutated. We used the Miniprep <a href="https://2012.igem.org/Team:Goettingen/week11-3">V07_12</a> of the liquid culture from the first mutagenesis round. It contains multiple plasmids that are mutated for the first three aa sites. The <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">PCR protocol</a> is basically the same as for the first round. </li> |
+ | </ul> | ||
+ | <ul> | ||
+ | <li>Observations & Results: <br>The corresponding agarose gel showed a strong band of the expected size (3769 bp). </li> | ||
</ul> | </ul> | ||
<br></td></tr> | <br></td></tr> | ||
</table> | </table> | ||
<br> | <br> | ||
- | + | <table cellpadding="20 px" border="1" bordercolor="black" valign="top"> | |
+ | <tr bordercolor="black" valign="top"> | ||
+ | <td width="900" bordercolor="black" valign="top"> | ||
+ | <h2><b>V07_17 </b></h2><br> | ||
+ | <b>V07_17_1 Test digestion of Miniprep V07_12 </b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>A test digestion using EcoRI and PstI in a 20 µL batch was performed according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> to confirm that the mutant plasmid pSB1C3_TAR_QC_18C has actually been isolated correctly. </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li>Observations & Results: <br>The corresponding agarose gel showed two distinctive bands of the expected sizes which led to the conclusion that the plasmid had indeed been isolated! </li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <b>V07_17_2 2<sup>nd</sup> round: Saturated mutagenesis PCR, volume 1 mL</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>The PCR was now set up in a volume of 1 mL to generate a high amount of DNA in order to reach the diversity we needed. The reaction was split to 20 50 µL tubes.</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | </td></tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | <table cellpadding="20 px" border="1" bordercolor="black" valign="top"> | ||
+ | <tr bordercolor="black" valign="top"> | ||
+ | <td width="900" bordercolor="black" valign="top"> | ||
+ | <h2><b>V07_19 </b></h2><br> | ||
+ | <b>V07_19_1 2<sup>nd</sup> round: PCR clean-up </b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>The clean-up was performed according to the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>. </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li>Observations & Results: <br>The corresponding gel showed bands of the expected sizes for each reaction tube. </li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <b>V07_19_2 2<sup>nd</sup> round: Digestion <i>Dpn</i>I/<i>Bsa</i>I and clean-up</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>The digestion and subsequent clean-up was performed according to the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>.</li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li>Observations & Results: <br>The corresponding gel showed bands of the expected sizes for each reaction tube. </li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <b>V07_19_3 2<sup>nd</sup> round: Ligation</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>The ligation was performed according to the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>. We set up a volume of 2 mL.</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | </td></tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | <table cellpadding="20 px" border="1" bordercolor="black" valign="top"> | ||
+ | <tr bordercolor="black" valign="top"> | ||
+ | <td width="900" bordercolor="black" valign="top"> | ||
+ | <h2><b>V07_20 </b></h2><br> | ||
+ | <b>V07_20_1 2<sup>nd</sup> round: Ethanol precipitation of ligation V07_19 </b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>The ethanol precipitaion was performed according to the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>. </li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <b>V07_20_2 2<sup>nd</sup> round: Transformation of electrocompetent cells with mutated plasmid mixture</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>The preparation of electrocompetent cells and the subsequent transformation were performed according to the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>. We adjusted the volume of liquid culture to 250 mL to reach diversity.</li> | ||
+ | </ul><br> | ||
+ | </td></tr> | ||
+ | </table> | ||
+ | <br> | ||
<a href="https://2012.igem.org/Team:Goettingen/Notebook">Back to overview</a><br> | <a href="https://2012.igem.org/Team:Goettingen/Notebook">Back to overview</a><br> | ||
<br> | <br> |
Latest revision as of 13:33, 25 September 2012
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#3 Chemoreceptor Library - 12th WeekBack to overview
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