31th August 2008

Wet Lab

Cloning

• Whenever the sequences from GeneArt arrive, priority becomes the rapid cloning of sequences into Biobricks to begin construction

Monday

• Test all primers for PCR cloning from pDR111 using taq to ascertain optimal consitions.

• If time allows, test genomic primers using B.subtilis chromosome sample 1 or using single colony PCR

Tuesday

• Carry out PCR cloning with Pfu, purify and clone DNA sequences into Biobricks

Wednesday

Test orientation of inserts to find a correct Biobrick containing host to culture overnight for midiprepping

Thursday - Friday

• Hopefully, construction begins...

B.subtilis

Monday

• Prepare all media and plates needed for the rest of the week,
• Carry out single colony PCR on B.subtilis transformed previously with pDR111 using verification primers. These verification primers work on the basis of flanking the chromosomal integration sites allowing the integration sites and any inserted elements to be amplified.

Tuesday

• Prepare overnight culture for the B.subtilis growth curve
• Perform a transformation using linear DNA derived from pDR110 or pDR111

Wednesday

• 2nd trial of the B.subtilis growth curve and OD₆₀₀ vs c.f.u. experiment
• Perform single colony PCR with the B.subtilis transformed yesterday using verifications primers.
• Begin to try to optimise the protocol for B.subtilis transformation protocol with linear DNA. This includes the volume of transformed cells, amount of DNA, the density of competent cells and length of DNA incubation.

Thursday

Friday

• Carry on to optimise the protocol for B.subtilis transformation protocol with linear DNA