26 August 2008

Wet Lab

B.subtilis

• The B.subtilis transformation protocol 1 , click here for the protocol, was not performed today.
• A 20ml B.subtilis culture was prepared in the afternoon. This culture will be used for the microscope tomorrow.
• In addition two E.coli 100ml cultures were grown overnight for midi preping. These E.coli contained the integration plasmids pDR110 and pDR111. We require these midi preps to PCR out biobricks and for use in transformation protocol optimisation.

Cloning

• Trial PCRs runs with a Pfu enzyme without endonuclease

Lanes : M = Marker, 1 = Vector test (-2°C), 2 = Vector test (-4°C), 3 = Vector test (-6°C), 4 = 100ng Genomic test (-2°C), 5 = 100ng Genomic test (-4°C), 6 = 100ng Genomic test (-6°C), 7 = 1μl Genomic test (-2°C), 8 = 1μl Genomic test (-4°C), 9 = 1μl Genomic test (-6°C), 10 = control (No template DNA)

• 40ng of vector was used for vector PCRs. Timings were the same as those for our Pfu protocol

• For the vector PCRs and the control, primers for the 5' AmyE integration sequence were used. For genomic PCRs, the XylR primers were used

• Only the vector PCRs were succesful and a contaminent was produced. Further trials will be required