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V08_20
V08_20_1 Attempt b: increasing the library diversity!
- Experiment:
A PCR (1000 µL) using the 2nd round primers (Tar6973 for and Tar6973 rev) and the liquid culture pellet Miniprep that was used at V07_25 as template was performed. Protocol according to week 10. By doing this and later combine the products, the diversity can be increased. Purification was carried our using the peqGOLD Cycle-pure Kit (PeqLab) with Miniprep Kit columns following the user manual.
- Observations & Results:
The corresponding agarose gel showed a band of the expected size.
V08_20_2 2nd round: Plasmid preparation from pellets #1-#4 V08_17
- Experiment:
peqGOLD Xchange Plasmid Midi Kit (PeqLab) was used following the user manual. Elution in 100 µL H2O. Stored at -20 °C.
- Observations & Results:
DNA concentrations were determined as follows using nanodrop:
#1 - 521 ng/µL
#2 - 1035 ng/µL
#3 - 2145 ng/µL
#4 - 1103 ng/µL
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V08_21
V08_21_1 Attempt b: DpnI/BsaI digest and subsequent purification
- Experiment:
The digest and purification was performed following the protocol from week 10.
- Observations & Results:
The corresponding agarose gel showed a band of the expected size.
V08_21_2 Attempt b: Ligation, 2000 µL
- Experiment:
The ligation was performed according to the protocol from week 10.
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V08_22
V08_22_1 Attempt b: Purification of ligation V08_21
- Experiment:
The purification was performed following the protocol from week 10.
- Observations & Results:
The corresponding agarose gel showed a band of the expected size.
V08_22_2 Attempt b: Transformation of electrocompetent cells with mutant plasmid mixture
- Experiment:
The preparation of electrocompetent cells was carried out according to protocol. The subsequent transformation was performed according to the protocol form week 10.
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V08_23
V08_23_1 Attempt b: Analysis of transformation plates and liquid culture V08_22
- Experiment:
Investigation of transformation.
- Observations & Results:
Worked.
V08_23_2 Transformation of E. coli BL21 and E. coli MG1655 with mutant library!
- Experiment:
Electrocompetent cells of E. coli BL21 and E. coli MG1655 were prepared according to protocol.
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V08_24
V08_24_1 Analysis of transformation plates V08_23
- Experiment:
Investigation of transformation.
- Observations & Results:
Worked. We have now successfully generated a mutant library consisting of 2x105 different mutants. For more information on the method and other results, please visit our "results" page. The members of group #3 now helped the other focus groups with their projects.
V08_24_2 Preparation of glycerol stocks
- Experiment:
Glycerol stocks (30 %) of E. coli BL21 and E. coli MG1655 were prepared according to protocol.
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