Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 6.29.08
I ran out the product from the gradient PCR looking for the optimum annealing temp for BT2736 and BT2737 using Taq and found that all annealing temps between 50 and 65 degrees worked. The global difference with this PCR is the 1 minute extension time (as opposed to the 50 second PCR I allowed for the previous PCR for the HmgR Orf).
I selected 7 colonies from the pIs001.3 transformation from Friday (plates removed and placed in fridge on Saturday morning), inoculated each in 50 μl H20, and then used 1 μl suspension in a 10 μl PCR. Will run out on a gel on Monday.
- Prepare sequencing file to compare with sequencing data when it comes back.
- Figure out all the details for the extraction protocol of HmgR from BL21.
- Run out colony PCRs on gel to see if any transformants have pIs001.3.
- If some colonies have the plasmid, set up cultures this evening for glycerol and miniprep.
I ran out the colony PCR products on a gel and found that colony 3 gave product. I started a 5 ml culture (5 μl Amp, 25 μl colony suspension) at 1715 for glycerol and miniprep tomorrow.
- See if pIs001 sequenced properly.
- If not, talk with Mayuree about going ahead with isolation.
- If it did, everything's good to go for tomorrow.
- Make a glycerol of colony 3 (transformed with pIs001.3) and do a miniprep.
- Submit prep of colony 3 for sequencing.
pIs001.2 looks pretty good. There are a few questionable areas (may not be a problem), but the major concern is that only ~540 bases were sequenced from the NdeI site (there are 804 bases in the ORF). I submitted more of the same prep to be sequenced with the reverse primer. I also finished prepping from the 6.25.08 transformation colony 3 culture and submitted 6 μl for sequencing with both the forward and reverse primers. As a note, the prep from colony 3 will be named pIs001.4. Assuming that both .2 and .4 sequence properly, I won't bother distinguishing between them (just call it pIs001).
I think it will be worth going ahead with the isolation tomorrow. Actually, go only so far as to get the pellets (before sonication) and put them in the -20. I'll rupture the cells and run on a gel the same day.
I also should use primers internal to the HmgR ORF to get some better reads at the ends. When I get good data on all regions, if there are any consistent mutations, check my source and then see if the mutation is in a wobble position (silent).
- Submit pIs001.2 and .4 for sequencing with BT1032 and BT1033.
- Do first part of isolation protocol (up to spinning down the induced pellet), then place in -20.
I completed the isolation protocol up to spinning down the cells after the 2 hour induction. I removed the supernatant (decanted and then used pipette to get residual fluid). I placed them in the -20 at 1530.
I'm going to be sending out some more sequencing tomorrow for pKn002 and pKn001, so I figured I wait to submit all of them at the same time.
- Submit pIs001.P1 and pIs001.P2 (formerly known as pIs001.2 and .4) for sequencing with BT1032 and BT1033 (and both pET primers for pIs001.P2).
- Do second part of isolation protocol
- Do a Bradford Assay on the fractions.
- Load and run the poly-ac gel
- Stain and destain the gel and image.
Tomorrow I need to get a protocol for the Heparin here. I also need to start an overnight of BL21 with pIs001 (5 ml), to grow up a 200 ml culture tomorrow. I can spin this down and freeze it and take some samples to run on an SDS-Page again to be sure my repressor behaves the same way when it's produced en masse.
- Use flask from wing B
- Don't bother doing a negative control tube - just take 1 ml of culture before induction and save it.
- Save a ml of culture after induction to run on gel.
- Complete En Masse HmgR Isolation Protocol up to the point of collecting the cells through centrifugation after induction with IPTG. Freeze the pellets at this point.
- Write the Heparin column purification protocol.
I completed the isolation protocol up to the induction - I spun down the culture, decanted the supernatant, and froze the pellets in the -20.