26 September 2008

Wet Lab

Cloning

• 5' and 3' AmyE integration sequences digested with XbaI and SpeI to check for insert orientation (incorrect insert oreintation destroys the XbaI and SpeI restriction sites)

U = undigested, D = digested

• Only one of the 5' AmyE minipreps cuts correctly so it is likely that this is the only correctly inserted gene. All three of the 3' AmyE minipreps were cut, however one insert was noticebly smaller. In particular, it appears to be the same size as the RFP-Terminator (positive control) and so it is probable that it is a contaminent bt the othe rtwo minipreps contain correctly orientated DNA.

• To further check integration, and to test for copying errors during PCR the midipreps that will be taken from these minis will require sequencing

SCP

• To confirm successful ligations of our parts into biobrick vectors we carried out single colony PCR (SCP). The gel below shows the results from the experiement. The lane notation is as follows:
  • 6L = LipA-HE
  • LacI - LacI gene
  • 12 = Pveg-spoVG
  • Pos = positive control
  • Neg = negative control

• The results show that LacI _{1 and 2} have worked and 12 ₃ have successfully ligated. None of the 6L constructs have successfully ligated.