Imperial College/22 September 2008

Wet Lab

Miniprep

• 9 minipreps were carried out for samples 5-1, 5-2, 8-1, 8-2, 8-3, 9-2, 9-3, 12-2 and 12-3.

Single Colony PCR

• A variety of ligations were carried out last week and to test there success we carried out single colony PCR using Psb primers to verify if an insert is present in the vector AK3. For each ligation thress seperate colonies were picked to carry out SCP and a negative colony where no DNA was present. The gel below shows the resuls of this experiment:

• The conditions used were as follows:
  • 1 cycle - 95^oC for 30 seconds
  • 30 cycles - 95^oC for 30 seconds, 60^oC for 30 seconds,72^oC for 30 seconds
  • 1 cycle - 72^oC for 2 minutes
• The numbers of the ligations correspond to the following ligation reactions:
  • 7-SacB-E+AK3
  • 8-LipA-E+AK3
  • 9-P43-gsiB+AK3
  • 10-P43-spoVG+AK3
  • 11-Pveg-gsiB+AK3
• Of the SCP the following look to have given a positive result:
  • 7_1,2,3
  • 8₁
  • 9_1,2
  • 10₃
  • 11-All too big
• 11 has failed and in addition there is a contamination within the negativ control. The next step is to repeat the SCP of the contruct 11