Team:Goettingen/week11-3

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Revision as of 18:15, 22 September 2012


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V07_10_1 1^st round: Digestion DpnI/BsaI and clean-up

Experiment:
The digestion and subsequent clean-up were carried out according to the protocol of week 10.

Observations and results:
The corresponding gel showed a band of the expected size.

V07_10_2 1^st round: Ligation

Experiment:
The ligation was carried out according to the protocol of week 10 and incubated over night at 16 °C.

V07_11_1 1^st round: Ethanol precipitation of ligated mutated plasmids

Experiment:
The ethanol precipitation was carried out according to protocol, this time with the right volumes!

Observations and results:
The corresponding gel showed a band of the expected size.

V07_11_2 1^st round: Transforamtion of electrocompetent cells wir the mutant plasmid mixture

Experiment:
Electrocompetent cells were prepared according to protocol. We transformed 200 µL of cells with 10 µL of DNA according to the protocol of week 10. Dilution plates and liquid culture was incubated over night at 37 °C, 180 rpm.

V07_12_1 1^st round: Analysis of transformation V07_11

Experiment:
Both the liquid culture and the dilution plates were checked for bacterial growth. 5 mL LB liquid cultures + CM were prepared for each clone on the plates for subsequent sequencing of the plasmids. The cultures were incubated over night at 37 °C and 180 rpm.

Observations and results:
Both the liquid culture and the dilution plates were positive for bacterial growth. Plates showed the following results:
10⁴ 10 clones
10⁵ 2 clones
10⁶ 0 clones

V07_12_2 1^st round: Miniprep of liquid culture V07_11

Experiment:
We used 4 x 5 mL liquid culture split to 4 columns for plasmid preparation with the peqGOLD Plasmid Miniprep Kit (PeqLab).

V07_13_1 1^st round: Miniprep of clones 1-12 V07_12

Experiment:
Plasmids from the 1^st mutation round were prepped using the peqGOLD Plasmid Miniprep Kit (PeqLab) following the user manual. DNA concentrations were determined for subsequent sequencing.

V07_13_2 1^st round: Sequencing

Experiment:
All 12 clones were sequenced using the primers VR, VF2 and two primers that bind inside tar labeled Seq01_TAR and Seq02_TAR. Here we aimed to examine whether a bias towards certain mutations was introduced.
Additionally we sequenced the generated mutagenesis library with primer VF2 in order to visualize the introduction of multiple mutated bases at the three predetermined sites.

Observations and results:
We were able to confirm that our mutagenesis technique did not introduce a bias towards certain mutations in the 1^st mutation round.
The sequencing chromatogram showed multiple overlaying bases at the predictged sites!

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 <ul>
 <li>Observations and results: <br>We were able to confirm that our mutagenesis technique did not introduce a bias towards certain mutations in the 1<sup>st</sup> mutation round.<br>
+The sequencing chromatogram showed multiple overlaying bases at the predictged sites!
 </li>
 </ul>