Team:Goettingen/week10-2

V07_02

• Experiment:
  In order to clone the flhDC-promoter constructs intro 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 purification of the desired fragments.


• Observations & Results:
  The gel did not feature fragments of the expected size.Obviously, something was wrong with the digestion.

• 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
  

V07_03

• Experiment:
  Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

• 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.

V07_04

• 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.

V07_05

• 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.

• Experiment:
  The test 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.

V07_06

• 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 old Pfu-polymerase was not functional anymore and had caused the PCRs failure.