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

7.7.08

To Do:

• When Mayuree has time, learn how to use the French Press to produce lysate from the pellets I've isolated (may be today or tomorrow).
• Write the Heparin column purification protocol today.
• Prepare Polyacrylamide Gel to run samples from 200 ml culture.
• Sonicate samples from 200 ml culture.
• Align sequence files for pIs001.P1 and pIs001.P2 when they arrive.

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:

• Write the Heparin column purification protocol.
• Prepare Polyacrylamide Gel to run samples from 200 ml culture.
• Align sequence files for pIs001.P1 and pIs001.P2 when they arrive.
• Come up with shortlist of DNA fragments to use in gel shift assay.

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.