Team:Goettingen/week14-3

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#3 Chemoreceptor Library - 14th Week

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V07_30

V07_30_1 2^nd round: Analysis of the transformation V07_27

Experiment:
The plates and liquid culture were analyzed.

Observations & Results:
Neither the liquid culture nor the plates exhibited bacterial growth.

V07_30_2 2^nd round: Saturated mutagenesis PCR, 1000 µL

Experiment:
The PCR was set up again, seeing as we lost our DNA material during the ethanol precipitation the previous week.

V07_31

V07_31_1 2^nd round: PCR clean-up

Experiment:
The PCR clean-up was performed according to the established protocol form week 10.

Observations & Results:
The corresponding agarose gel showed a band of the expected size.

V07_31_2 2^nd round: DpnI/BsaI digestion and purification

Experiment:
The digestion was performed with a total volume of 300 µL according to protocol of week 10. The purification was now again performed using the peqGOLD Cycle-pure Kit (PeqLab). This should stop the loss of DNA material throughout the purification steps!

Observations & Results:
The corresponding agarose gel showed a band of the expected size.

V07_31_3 2^nd round: Ligation

Experiment:
The ligation was set up in a 100 µL batch according to protocol. Incubation over night at 16 °C.

V08_01

V08_01_1 2^nd round: Ethanol precipitation if ligation V07_31

Experiment:
The ethanol precipitation was performed according to the established protocol form week 10.

Observations & Results:
The corresponding agarose gel showed a band of the expected size.

V08_01_2 2^nd round: Transformation of electrocompetent cells with mutant plasmid mixture

Experiment:
Electrocompetent cells were prepared according to protocol. The transformation was performed according to protocol of week 10.

V08_02

V08_02_1 2^nd round: Analysis of transformation V08_01

Experiment:
Plates and liquid culture were checked for successful transformation.

Observations & Results:
No bacterial growth on neither plates nor in liquid culture. Again, our DNA material was presumably lost during the ethanol precipitation. Confirmation see V08__02_2!

V08_02_2 2^nd round: Test digestion of the ethanol precipitation samples

Experiment:
A test digestion with EcoRI and PstI was performed on the samples from V08_01_1.

Observations & Results:
The corresponding agarose gel did not show any bands! There was no DNA material present in the ethanol precipitated samples.

V08_02_3 2^nd round: mutagenesis PCR from V07_30 repeated

Experiment:
The PCR was performed according to the established protocol from week 10.

V08_03

V08_03_1 2^st round: PCR clean-up

Experiment:
The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) following the user manual. We modified the protocol by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) because these can bind a higher amount of DNA (30 µg/column). Thus, we lose fewer DNA material in the cleaning steps. 50 µL of elution buffer was used.

Observations & Results:
The corresponding gel showed a band of the expected size.

V08_03_2 2^nd round: DpnI/BsaI digestion

Experiment:
The digestion was performed with a total volume of 200 µL according to protocol.

V08_03_3 2^nd round: Digestion clean-up

Experiment:
The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) modified by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) with an EB volume of 100 µL.

Observations & Results:
The corresponding gel showed a band of the expected size.

V08_05

2^nd round: Ligation

Experiment:
The ligation was set up in a 2000 µL batch according to protocol. Incubation over night at 16 °C.

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@@ Line 60: / Line 60: @@
 </ul>
 <br>
-<b>V07_31_3 1<sup>st</sup> round: Ligation</b><br>
+<b>V07_31_3 2<sup>nd</sup> round: Ligation</b><br>
 <ul>
 <li>Experiment: <br>The ligation was set up in a 100 µL batch according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. Incubation over night at 16 °C.
@@ Line 129: / Line 129: @@
 </ul>
 <br>
-<b>V08_03_2 2<sup>st</sup> round: <i>Dpn</i>I/<i>Bsa</i>I digestion</b><br>
+<b>V08_03_2 2<sup>nd</sup> round: <i>Dpn</i>I/<i>Bsa</i>I digestion</b><br>
 <ul>
 <li>Experiment: <br>The digestion was performed with a total volume of 200 µL according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.
 </li>
 </ul><br>
-<b>V08_03_3 2<sup>st</sup> round: Digestion clean-up</b><br>
+<b>V08_03_3 2<sup>nd</sup> round: Digestion clean-up</b><br>
 <ul>
 <li>Experiment: <br>The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) modified by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) with an EB volume of 100 µL.
@@ Line 149: / Line 149: @@
 <td width="900" bordercolor="black" valign="top">
 <h2><b>V08_05 </b></h2><br>
-<b>2<sup>st</sup> round: Ligation</b><br>
+<b>2<sup>nd</sup> round: Ligation</b><br>
 <ul>
 <li>Experiment: <br>The ligation was set up in a 2000 µL batch according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. Incubation over night at 16 °C.
@@ Line 176: / Line 176: @@
 /li>
 b>V07_31_2 2
+/li>