Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 7.27.08

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To Do:

  • Send prep pKn004.P2 from second pKn004.L3 clone for sequencing.
  • Obtain the Lox/Antibiotic cassette for ligation into both pKn003 and pKn004.


I sent off the prep for sequencing and I got the Lox/Gentamicin Cassette (pCM351) from P'So. I need to digest and ligate into pKn003 and pKn004 tomorrow, then transform into DH5 α.


To Do:

  • Digest Lox/Gentamicin cassette with the appropriate enzymes, purify, and ligate into pKn003 and pKn004.
  • Transform pKn005 and pKn006 into DH5α.

Thinking about best enzymes to use for digestion of pCM351 for ligation into pKn003 and 4. Mayuree's plan calls for cutting 003 with BamHI or XbaI and 004 with SphI or SacII. Ideally I could cut with 2 enzymes (I don't think I can get the cassette out of pCM351 with one enyzme) and be able to ligate into 004 and 5 without blunt ending.

Neither BamHI nor XbaI shares complementary overhangs with any of the cloning sites on pCM351, but there is an XbaI site.

There is a SacII site on pCM351 as well as pKn004, but no other complementary overhangs for SacII. No complementary overhangs for SphI.

It looks like blunt ending is unavoidable. For HmgR (004), I'm going to use SphI so that if I need to excise the downstream fragment I just ligated in, I can do so without having to remove the cassette as well by using the SacII site.


Digested pKn003 (see notes on digest here), pKn004 (see notes on digest here), and pCM351 (see notes on digest here) and ran the last out on a gel and excised. I will do the purification, blunt ending, ligations, and transformation tomorrow.


To Do:

  • Gel purify Lox/Gen cassette
  • Blunt end pKn003/4 and Lox/Gen cassette.
  • Ligate the cassette into either vector.
  • Transform into DH5α.


I blunted each of the vectors and the cassette after gel purifying the latter. I also did the ligations and transformations into DH5α. Will do colony PCRs tomorrow. Can use M13_rev and BT297 or M13_for for pKn005 (I think I found an error in my annotation - BT2724 should have been removed after the NcoI digest). BT1188 and M13_for should work for pKn006.


To Do:

  • Colony PCR of pKn006 candidate.
    • If it looks promising, set up overnight for glycerol and prep.
  • Try ligations again with higher insert/vector ratios.


To Do:


I did the new ligation and transformed. Hopefully some good news tomorrow.



Transformation only yielded products on the pKn006.L2 pellet plate again. I will select the colonies on Monday and do PCRs. I need to think about the other construct though.

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