Team:British Columbia/Notebook/Week 5

3-Carene

Daisy has been trying to figure out why the sequencing did not work. It may be due to the miniprep method. She will continue to make the yeast construct and if the sites are still there, she can always do another mutagenesis on the yeast plasmid.

1,8-Cineole

Jacob ran a restriction digest and gel to check his variant SDM-PCR product. It was successful! However, his attempt to remove another restriction site from pg-tps-cin did not work so Jacob ran another SDM with more PFU - it worked! Jacob then transformed E. coli with this product. Using plasmid mini-prepped from successful transformants, Jacob proceeded to SDM the next restriction site...

... which also worked! Now, both restriction sites from pgxe-tps-cin and 2 of 3 restriction sites from pg-tps-cin have been successfully removed. One more to go! Jacob set up the (hopefully) final SDM to run overnight...

Success! The gel showed that the SDM was successful and another gel, which showed a restriction digest of the previous SDM products suggests that the SDMs were all successful. Jacob then did the final transformation with the fully SDMed products.

Beta-pinene & (-)-limonene

Marianne repeated her SDM of beta-pinene by using both Pfu and Taq. Gel verification showed a smear so she decided to run a gel on the mini-prep of the beta-pinene plasmid from Rafael to make sure that the DNA wasn't contaminated... Fortunately, there were bands at the correct sizes indicating that the plasmid is fine. So she repeated the SDM using temperature-ramping...

Finally! Gel verification of the SDM beta-pinene product shows bands of the correct size suggesting that the SDM was successful. This was transformed into E. coli.

However, the SDM limonene product appeared as smears and various bands, suggesting unspecified annealing of primers. More troubleshooting: DMSO could be lowering the Tm of the primers; repeat without adding DMSO for one sample, without adding MgCl₂ for another sample, use the ramping cycling condition previously indicated. However, even after these modifications, further SDMs did not yield the desired product... Vicki is very frustrated!

Extra Parts: IDI1, HMG2, erg20-2

We received and amplified 5 plasmids from Greece containing various parts and synthase genes: IDI1, HMG2, p300, Cin, Sab. These plasmids were transformed into yeast to make sure they were in working order.

Still awaiting the erg20-2 part from France...