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Team:British Columbia/Notebook/Week 6
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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. | 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. | ||
Revision as of 03:27, 4 August 2011
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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.
