Team:British Columbia/Notebook/Week 6

Beta-pinene

All 3 transformation plates (P2SDM1, P2SDM2, P2SDM3) have colonies! Marianne set up overnight cultures, mini-prepped them and sent them for sequencing. Hopefully the SDM worked and the beta-pinene synthase gene no longer has illegal cut sites.

(-)-limonene

SDMs have failed thus far. More troubleshooting: use less pfu as the glycerol content in the enzyme could alter the reaction - prepared 3 samples, each with 0.5uL pfu, 1ul pfu, and 2ul pfu, respectively. Also prepare a sample using 0.5 taq polymerase only. Also increased the annealing temperature and elongation time.

Again, gel verification shows no bands. More troubleshooting: use a different cycling condition.

Again, no bands. Something is amiss...

1,8-cineole

After 5 SDMs, transformations and minipreps, Jacob should now have ECORI-less, SPEI-less, XBAI-less, PSTI-less pg-tps-cin and pgxe-tps-cin. The gel of the restriction digest has the correct number of bands. Sequencing is the next step to confirm this result.