Team:Goettingen/week19-2

V09_03

• Experiment:
  In order to eliminate undesired restriction sites inside of our part a Quikchange PCR was performed using especially designed primers that will cause a point mutation and Pfu Turbo polymerase (see Quick Change protocol in "protocols"). Since four unwanted restriction sites are localized in our part four reactions were prepared producing overlapping fragments. Subsequently samples 1 and 2 as well as 3 and 4 were combined via overlapping PCR reducing the number of fragments at two. Finally smaple 1+2 and 3+4 were combined as well and one final DNA fragment is obtained.
  
• Observations & Results:
  The QC PCR was not successful. We were not able to observe any PCR product.

V09_04

• Experiment:
  The test digestion of pSB1C3 including flHDC under the expression of 8 different promoters was performed with the restriction enzymes EcoRI and PstI according to the protocol. The product was subsequently analyzed on an 1% analytical agarose gel.
  
• Observations & Results:
  Since fragments of the expected size could be obtained for all clones the test digestion worked for all.

• Experiment:
  PCR of motA, motB and yhjH using primers containing the standard BioBrick sequences. Procedure was succesful and the PCR products were purified via PeqGOLD Gelextraction Kit (Peqlab).

V09_05

• Experiment:
  motA, motB, yhjH and pSB1C3 were digested with EcoRI and PstI. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).

• Experiment:
  Here again the Quikchange PCR was performed using designed primers and Pfu Turbo polymerase (see Quick Change protocol in "protocols"). However, this time a lower annealing temperature was chosen. Also this time four short fragments were produced first and subsequently combined to a complete final DNA fragment.
  
• Observations & Results:
  Once again the QC PCR failed for no PCR product was observed. Since we already had problems with enzymes during our experiments we consider an non-functionable polymerase.

V09_06

• Experiment:
  The ligation of motA, motB and yhjH with pSB1C3 was conducted as described in the protocol.
  

• Experiment:
  The transformation was performed as described in the standard protocol.
  
• Observations & Results:
  The transformation was successful since numerous colonies has formed whereas the negative control remained clear.

V09_09

• Experiment:
  Over night cultures were prepared:
  pSB1C3-motA
  pSB1C3-motB