Team:Goettingen/week11-2

V07_09

• Experiment:
  The amplicons were separated via application on an 1% agarose gel and subsequently cut out. The DNA was purified out of the gel slices using peqGOLD Gel Extraction Kit (Peqlab) according to the user manual.
  

V07_10

• Experiment:
  In order to clone the new genes into pUC18, all components were digested with EcoRI and XbaI according to the protocol. The restricted plasmid pUC18 was subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. The other digestion products were investigated via preparation of an 1% analytical gel and directly purified using the peqGOLD Gel Extraction Kit (Peqlab) according to the manual as well.
• Observations & Results:
  The digestion was successful. For all PCR products as well as for the vector band of the expected size could be obtained and the fragments can now be used for further cloning.

• Experiment:
  The ligation of the digested genes with the plasmid pUC18 was conducted as described in the protocol.

V07_11

• Experiment:
  For the chemical transformation the standard protocol was followed.
  
• Observations & Results:
  The transformation was successful since all plates showed numeours colonoes except the negative control.

V07_12

• Experiment:
  Over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in pUC18 were prepared in order to isolate the plasmids.
  

V07_13

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

• Experiment:
  All vectors were digested with EcoRI and XbaI according to the protocol. The restriction products were subsequently separated on a gel in order to determine successfully ligated plasmids.
  
• Observations & Results:
  Except for fliC (Salmonella) #1 all picked colonies seem to host plasmids containing the aimed insert.

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.