Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 6.16.08

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6.16.08

The primers BT2736 and BT2737 arrived today. I added 277 and 315 μl of TE to them respectively to create 100 μM Stocks. I then diluted each 1 μl in 99 μl H20 to have my own 1 μM stocks. I placed the TE stocks in the -20 in the latest box and I put my aliquots in my -20.

Now that I have the primers, I can plan to do the PCR. Here's the protocol. I should check if I'm still using Pfu (I think so).

6.17.08

To Do:

  • Run the PCR to amplify the HmgR ORF. If the other PCR worked, I can run both the Upstream fragment and HmgR amplification together because they both use the same annealing temp (55o).
  • Assuming the HmgR ORF PCR works, digest with NdeI and BamHI. I might as well run out on a gel to get rid of unwanted fragments.
  • Get pET-11a and digest with NdeI and BamHI to prepare for insertion of HmgR ORF.
  • Do a gel clean up of the excised bands.

6.18.08

To Do:

  • Try a gradient PCR for the annealing temperature for the amplification of the HmgR ORF.
    • What should be the temperature bounds? Try 50oC to 65oC (5 degrees below the annealing temp of the lower primer to well above the annealing temp of the higher primer).
  • Run the gradient PCR products on a gel to see if any products show up.
    • If bands show up, note the temperature and rerun the PCR with a larger volume at the best temperature (I can further optimize if I so choose).
    • If no bands show up, I should probably repeat the gradient PCR as my run was a little abnormal :-).


Summary:

The gradient PCR worked - actually, each annealing temperature worked and it doesn't seem that there's a particularly efficient temperature between 50 and 65 degrees. I excised the bands (8 of them with 5 μl of product in each lane) and did a gel clean up, capturing everything on one column. Tomorrow I'll try digesting.

6.19.08

To Do:

  • Digest gel-extracted PCR product with NdeI and BamHI
  • Run out digested product on a gel, excise, gel purify.
  • Ligate digested product into pET-11a (already digested).
  • Transform into some cloning strain?
    • If the pET-11a doesn't allow for the Blue-White selection, how will I go about selecting colonies with the insert? Screening?

Summary:

I forgot I had the cultural orientation today, so everything is postponed until Friday.

6.20.08

To Do:

  • Digest gel-extracted PCR product with NdeI and BamHI
  • Run out digested product on a gel, excise, gel purify.
  • Ligate digested product into pET-11a (already digested).
  • Transform into DH5 α.
    • If the pET-11a doesn't allow for the Blue-White selection, how will I go about selecting colonies with the insert? Screening?

Summary:

Digestion, gel, and ligation complete. Also finished transformation. Placed plates in 37o incubator at 16:45.

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