Team:British Columbia/Notebook/Week 5

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Contents

July 04 2011

IDI1, HMG2, erg20-2

  • Validated competent cells by transforming with pet100 vector containing Cineole synthase. They are in working order.
  • Amplified 5 plasmids received from Greece (IDI1, HMG2, p300, Cin, Sab). NOTE: did not properly centrifuge during DNA precipitation stage. Will validate plasmids in yeast transformations to make sure plasmids are in working order.
  • Awaiting materials from France (erg20-2).

1,8-Cineole

  • Ran a gel to test restriction digest on variant SDM-PCR product. Looks like the SDM was successful!

beta-pinene & (-)-limonene

  • SDM beta-pinene again by using both Pfu and Taq.
  • Gel verification shows a smear.
  • Troubleshooting: run a gel on the miniprep of the beta-pinene plasmid received from Rafael to make sure that the DNA hasn't been contaminated/digested.

July 05 2011

beta-pinene & (-)-limonene

  • Ran a gel on the miniprep of beta-pinene and (-)-limonene (SDM template).
  • Shows bands at the correct sizes indicating that the plasmid is present.
  • SDM beta-pinene and (-)-limonene again using ramping (ramping rate of 0.2 degrees C/sec).

1,8 Cineole

  • Jacob tried to remove another restriction site from pg-tps-cin. The SDM did not work.

July 06 2011

beta-pinene

  • Gel verification of the SDM product shows bands of the correct size suggesting that the SDM was successful.

(-)-limonene

  • Gel verification of the SDM product shows some lanes with smears, and another with 2 bands suggesting unspecified annealing of primers. SDM failed.
  • Troubleshooting: DMSO could be lowering the Tm of the primers; Repeat the SDM PCR without adding DMSO for one sample, without adding MgCl2 for another sample. Use the ramping cycling condition previously indicated.
    • Gel verification of the SDM product shows no bands. Addition of DMSO and MgCl2 is not the problem.

1,8-Cineole

  • Ran another SDM on pg-tps-cin, this time with more PFU. The gel suggests that it worked! Jacob then transformed E. coli with this product.

July 07 2011

beta-pinene

  • Transformed the SDM products.

1,8-cineole

  • Colonies! The product from the SDM to remove a restriction site from pgxe-tps-cin successfully transformed E. coli. On to the next restriction site!

July 08 2011

1,8-cineole

Both restriction sites from pgxe-tps-cin and two of the three restriction sites from pg-tps-cin have been successfully removed using SDM. One more to go! Jacob set up the (hopefully) final SDM to run overnight.

July 09 2011

1,8-cineole

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 are working. Jacob then did the final transformation with the fully SDM'd products.

Ampicillin Plates

The team was running low on ampicillin plates, so Jacob made more and will test them soon.

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