David Roche 10:31, 21 August 2009 (EDT)
WaaL Ligase PCR
2nd attempt, as it did not work yesterday
PCR was using:
- TOP10 e-coli genome.
- Pfu II Ultra Enzyme
- Pfu II Ultra Buffer
- dNTPs, fwd and reverse primers from freezer, water.
PCR did not give results when run on a gel. Primers showed, but no DNA. Same results as yesterday. Will try again with Barnes buffer replacing the Pfu II Ultra Buffer.
Gel Extraction of OtsA, RcsB, Dam
After the restriction digests of OtsA, RcsB and Dam from yesterday, we excised the genes from the gel they were run on. gel extraction was then performed on these fragments to yield the pure DNA.
Next Step : Run these genes and yesterday's OtsB digest against the vector, to give a comparison between the ratios of each needed for the ligation reaction.
Completed mini on 3 BioBricks - gave the DNA to Dineka to do a digest & run products on gel. This will confirm whether the BioBricks were successfully transformed into the chassis.
Plated out cells for chemical induction of colanic acid. Will leave on bench over the weekend to see if we get anything on Monday.