Team:Goettingen/week13-2

V07_23

• Experiment:
  New over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in DH10B, BL21, DH5alpha and XL1 Blue were prepared.

V07_25

• Experiment:
  LB medium containing ampicillin was inoculated with the over night culture and shaken until an optical density of 0.6-0.8 was reached. Meanwhile ampicillin containing M9 agar plates were prepared and autoclaved Whatman Filter Papers were imbued with tryptone solution. Then the cultures were dropped on the agar at a distance of 1 cm to the paper. On each plate also colonies hosting the empty vector pUC18 were dropped in order to have a reference. Two drops were rather applied into the agar than onto as done before, because some paper predicted faster motility. Also this time each plate was prepared in duplicates.
  
• Observations & Results:
  Here again a certain diversity in the growth rate could be observed. Whereas some cultures had already reached and optical density of 0.8, others were still at merely 0.04.Here again fliC featured a low density, this time in all strains and DH10B and BL21 show a general decreased growth rate. Striking is the fact that the strains that featured the best motility as well as the construct that seems to have the biggest influence show decreased division rate. Whether this phenomenon is a coincidence or not we are not sure about yet. At the following day (26th of July) as well as the day after (27th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed.

V07_02

• 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.

• 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.