Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 7.6.08

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7.7.08

To Do:

Summary:

Went home a bit sick today, everything postponed until tomorrow. Mayuree will help me with the French Press on Friday. Also, no need to sonicate cell samples - just boil in loading dye.

7.8.08

To Do:

Summary:

I ran the Polyacrylamide Gel with cell lysate from before and after induction of the 200 ml culture and found that my protein is still expressed in mass. I need only confirm that the sequencing is OK and then everything will be go for the isolation and subsequent binding assay (likely to be fluorescence anisotropy).

7.9.08

To Do:

  • Image the gel from yesterday.
  • Prepare 2 Polyacrylamide Gels for Jim's class and for my isolation on Friday.
  • Write the Heparin column purification protocol.
  • Clean the column I'll use for the purification.
  • Come up with shortlist of DNA fragments to use in gel shift assay.
    • Look for HmgR and GpxR boxes (putative boxes from Mayuree) at the HmgA/HmgR promoter (they're divergently transcribed, remember).
    • Maybe write a script to help me find the boxes.
  • Align sequence files for pIs001.P1 and pIs001.P2 when they arrive.

Summary:

Imaged the gel. Prepared poly-ac gels. Looks like the column soaking worked. Tomorrow I need to finish writing the heparin protocol.

7.10.08

To Do:

  • Write the Heparin column purification protocol.
  • Come up with shortlist of DNA fragments to use in gel shift assay.
    • Look for HmgR and GpxR boxes (putative boxes from Mayuree) at the HmgA/HmgR promoter (they're divergently transcribed, remember).
    • Maybe write a script to help me find the boxes.
  • Align sequence files for pIs001.P1 and pIs001.P2 when they arrive.

7.11.08

To Do:

  • Come up with shortlist of DNA fragments to use in gel shift assay.
    • Look for HmgR and GpxR boxes (putative boxes from Mayuree) at the HmgA/HmgR promoter (they're divergently transcribed, remember).
    • Maybe write a script to help me find the boxes.
  • Align sequence files for pIs001.P1 and pIs001.P2 when they arrive.

Summary:

Mayuree supervised me in lysing the cells and running the lysate on a column. I used the French Press, but it wasn't working properly so I used the sonicator, spun down the cells (10,000 for 5 minutes), prepared the heparin-sepharose column and ran the lysate on it, then eluted the protein in 28 fractions. I used a Bradford (1 in 2 dilution of 5x stock) to assay the fractions with the most protein and ran them on poly-ac gels . I dialyzed the protein and placed on ice for the weekend. Lots of stuff and I didn't take terribly good notes. I'll do my best to record what I did under HmgR purification.