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V07_02
V07_02_1 Preparative double digestion of 20E-flhDC, 2G-flhDC, 18O-flhDC and pSB1C3
- Experiment:
In order to clone the flhDC-promoter constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments.
- Observations & Results:
The gel did not feature fragments of the expected size. Obviously, something was wrong with the digestion.
V07_02_2 Preparation of over night cultures
- Experiment:
In order to gain more plasmid material of the flhDC-promoter constructs over night cultured were prepared for a subsequent plasmid isolation.
20E - #2
20G - #1
2G - #1
20I - #2
18M - #2
18O - #2
18K - #1
18C - #2
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V07_03
V07_03_1 Miniprep of the flhDC-promoter constructs
- Experiment:
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.
V07_03_2 Amplification of the genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH
- Experiment:
The genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH were amplified by pfu polymerase according to the following protocol. The success of the PCR was subsequently investigated preparing a 1% agarose gel.
- Observations & Results:
The PCR failed for no bands were visible on the gel whereas the marker was clearly discernible.
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V07_04
Repetition of the amplification of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH
- Experiment:
The genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH were amplified by pfu polymerase according to the following protocol. Additionally, a negative control as well as a positive control were prepared this time. The success of the PCR was subsequently investigated preparing a 1% agarose gel.
- Observations & Results:
The did not function again for no bands were visible on the gel; only the marker was clearly discernible. However, since also the positive control did not feature the slightest band, we assume that something might be wrong with our polymerase.
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V07_05
V07_05_1 Repetition of the amplification of the genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH
- Experiment:
To give it a last try the fliC (DH10B), fliC (Salmonella), motA, motB and yhjH were amplified by pfu polymerase according to the following protocol one last time. Here again, a 1% agarose gel was prepared to investigate the PCRs outcome.
- Observations & Results:
The gel of the PCR delivered the same picture as last time. No band could be observed except for the marker. Even the positive control does not work. Thus, we decided to buy a new polymerase.
V07_05_2 Repetition of the preparative double digestion of 20G-flhDC, 20I-flhDC, 18K-flhDC, and 18C-flhDC
- Experiment:
The digestion of the flhDC-promoter constructs EcoRI and PstI were applied according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments.
- Observations & Results:
The digestion was successful. Bands of the expected size could be observed and cut out.
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V07_06
Repetition of the amplification of the fliC (DH10B), fliC (Salmonella), motA, motB and yhjH using the new polymerase
- Experiment:
The genes fliC (DH10B), fliC (Salmonella), motA, motB and yhjH were amplified by Phusion polymerase according to the following protocol. We also prepared a negative control as well as a positive control. The success of the PCR was subsequently investigated preparing a 1% analytical agarose gel.
- Observations & Results:
Finally,the PCR was successful! We were able to receive bands of the expected size for each construct. Obviously, the pfu polymerase was not functional anymore and had caused the PCRs failure.
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