Matt Gethers/CRI, Thailand/Labwork/HmgR Binding Assay/Week of 7.20.08

7.21.08

To Do:

*Quantify the amount of isolated HmgR with a Bradford.

*Run HmgR and GpxR out on a gel - look for purity.

• Review all sequencing data (some wasn't very readable) for any major concerns.

*Talk with Mayuree about the probe.

Summary:

I did a Bradford on the purified HmgR and found that concentration of protein is 2.545 g/L or ~90.5 μM. I loaded a poly-ac gel with 1 and 2 μl of it so I have approximately 2.5 and 5 μg respectively. I also loaded some of P'So's GpxR.

Looking at the sequencing data, several things are apparent. First, the reverse primers for pIs001.P1 failed miserably. Second, BT1033 appears to be sequencing in a completely wrong place (not in between forward and reverse primers), third, BT1032 just doesn't seem to be working at all.

7.22.08

To Do:

• Show Mayuree gel - pure enough?
  • Clean the coomassie film off of the gel and use the scanner to quantify the peaks in each lane - purity?
  • Ask Ji to hook up the computer to the scanner.
  • Ask P'So how much GpxR she has.

*Ask Mayuree about the FA experiment; what do I have to do to be prepared when the probe comes?

*Ask Mayuree about information on GpxR - I can't seem to find it anywhere.

• Finish aligning sequencing data - be sure I have all the files organized properly.

Summary:

I asked Mayuree for materials on the FA binding experiment we'll be doing when the probe comes. I'm starting a protocol here. I'll likely also include notes on data analysis there for now. I still need to use the gel scanner to quantify the purity of the bands. I started, but still need to finish analyzing sequence data.

7.23.08

To Do:

• Finish aligning sequencing data - be sure I have all the files organized properly.
• Show Mayuree gel - pure enough?
  • Clean the coomassie film off of the gel.
  • Ask Ji to hook up the computer to the scanner.
  • Use the scanner to quantify the peaks in each lane - purity?
  • Ask P'So how much GpxR she has.
• Write protocol for FA experiment.
• Streak out 1st and 2nd clones carrying pIs001.
• Amplify HmgR again using reliable polymerase - need to begin cloning and purification process again.

Summary:

I've about finished looking through sequencing data for both clones transformed with pIs001. The first clone (the one used to isolate HmgR) looks to have an insertion, so I'm a little worried. The second clone has a really clean read from the forward primer, so I need to finish looking at the reverse primer. In any case, I've streaked out both of these clones on LB Amp plates for sequencing/transformation into BL21 and I began a PCR with a new polymerase that will hopefully give me high fidelity cloning. Note that I used 'colony #1' of the glycerols I made on 6/27 and the glycerol from 7/1 to streak out the plates.

I still need to find Ji at a convenient time so I can quantify the purity of my HmgR, but given the sequence data, that may be less of a priority now :-(

7.24.08

The plates I streaked from clones 1 and 2 grew up Ok (they were a bit dense for my liking, though).

To Do:

• Grow up cultures of clones 1 and 2 of pIs001, prep, and resubmit for sequencing with forward and reverse primers.
• Look at regions not covered by alignment in sequencing data.
• Use the scanner to quantify the peaks in each lane - purity?
  • Ask Ji for help.
• Ask P'So how much GpxR she has.
• Write protocol for FA experiment.

Summary:

I started a 5 ml culture (10 ml LB, 10 μl amp, split in two) for each clone and put in the warm room at 0845, but by 1600 they weren't very turbid. I'm starting overnights (same make up as this morning) as well as leaving the current ones in the shaker. Overnights in at 1630.

7.25.08

To Do:

• If cultures of clones 1 and 2 of pIs001 grew up, mini-prep and resubmit for sequencing with forward and reverse primers.
• Use the scanner to quantify the peaks in each lane - purity?
  • Ask Ji for help.
• Ask P'So how much GpxR she has.
• Write protocol for FA experiment.

Summary:

Both the cultures I started yesterday morning and the overnights of the 1st and second clones of pIs001 grew up. I miniprepped the Overnights, as I figure these have less of a chance of mutating with the shorter time in the incubator. I've designated the preps pIs001.P3 and pIs001.P4 and noted their origins. I've submitted for sequencing 6 μl each (forward and reverse).