Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 7.20.08

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7.21.08

To Do:

*Screen a second round of colonies from the pKn003.L2 transformation for more candidates.

*Find controls for SphI and HindIII, digest, and run out on gels. See if any cutter isn't working properly.

*Prepare sequence file for pKn003.P1 and annotate in OWW.

  • Align and review sequence data pertaining to mutant construction thus far.

Summary:

I selected 12 more colonies from last Monday's transformation and did colony PCRs. I found that one colony, #7, gave a product that looked about correct. I started a 5 ml overnight culture to make a glycerol and miniprep tomorrow.

I also did another round of digesting with SphI and HindIII, this time with some controls and trying a serial digest. I'll run the products out on a gel tomorrow.

7.22.08

To Do:

  • Run out digestion products on gel - see if there's any indication that the serial digest works.
  • Depending on digest results, ligate fragment 4 into pKn002.P1 again.
  • Transform ligation product (pKn004.L3) into DH5α.
  • Finish examining sequence data.

Summary:

I ran out the digest product on a gel and it looks like both enzymes are functioning properly, but the experiment I did can't really tell me if the double or serial digest worked. In case I'm getting any doubly digested vector, I'm treating an aliquot of the vector with phosphotase - I hope this will lower the frequency of self-ligation. I'm not transforming tonight - I'll do that tomorrow afternoon.

7.23.08

To Do:

  • Finish examining sequence data.
  • Transform ligation product (pKn004.L3) into DH5α.

Summary:

I transformed pKn004.L3 and pKn004.L4 into DH5α. Will select colonies and run colony PCRs tomorrow. Didn't get to sequencing data yet.

7.24.08

To Do:

  • Do colony PCRs on the pKn004.L3 and L4 transformants and run out on gel.
  • If colonies yield correct product, start overnight cultures.

Summary:

The colony PCRs yielded one good candidate - colony 6. I started an overnight culture (5 ml LB, 5 μl Amp, 25 μl colony suspension). Will make a glycerol and prep tomorrow as well as send off for sequencing. As I only found one, I'm going to do a second round of colony PCRs only on pKn004.L3 colonies - 12 of them.

7.25.08

To Do:

  • Run yesterday evening's colony PCRs out on a gel - look for more clones.
    • If any more clones, start overnight cultures for glycerol and prep tomorrow.
  • Make glycerol and miniprep overnight culture started for pKn004.L3 clone I found yesterday. Submit for sequencing.
  • Review sequencing from pKn003.
    • If no problems, plan to insert cassette to produce pKn005.

Summary:

I found a second clone from the second batch of colony PCRs. At 1430 I started an overnight for a glycerol and prep tomorrow. I made a glycerol and prep of the clone I found yesterday and submitted for sequencing this afternoon. The sequencing from pKn003 looks good with what's available, but there are large unsequenced regions I need to get to. I should identify some internal primers that can be used and use them for sequencing. Alternatively, I can wait until I add the Lox cassette before sequencing one final time.

7.26.08

To Do:

  • Make glycerol and prep from second pKn004.L3 clone.

Summary:

Made glycerol and prep. Must submit for sequencing on Monday.

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