Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 8.3.08

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8.4.08

To Do:

  • Do colony PCR for putative pKn006.L2 transformants.
  • Rethink cloning for pKn005.
  • Submit pKn006.P1 for sequencing.
  • Get a protocol for transformation into pseudomonas and transform pKn006 into it.

Summary:

Got an electroporation protocol from p'So. Will talk with Mayuree and hopefully start it tomorrow evening.

8.5.08

To Do:

  • Do colony PCR for putative pKn006.L2 transformants.
  • Rethink cloning for pKn005.
  • Submit pKn006.P1 for sequencing.
  • Transform pKn006 into Paeru.

Summary:

Completed colony PCR and found all clones looked about right, but some seem to have run slightly farther down the gel than others. I took a representative sample (2 colonies) and inoculated overnights - master of 10 ml LB, 10 μl amp, 10 μl Gen, split in two. 10 μl colony suspension. In at 1700.

8.6.08

To Do:

  • Make glycerols and preps of pKn006.L2 cultures I grew up last night.
  • Try ligating the cassette into pKn003 again to produce pKn005.
  • Set up overnight of PA01 for electroporation tomorrow.

Summary:

I made glycerols of the pKn006.L2 transformants and made preps: pKn006.P2 and P3. Need to submit for sequencing along with P1 today. Started 18 ml overnight culture of PAO1 started from old plate (18 ml LB,no antibiotic). Also streaked out new plate of PA01 from glycerol (LB-Agar, no antibiotic).

8.7.08

To Do:

  • Submit pKn006 preps for sequencing
  • Transformation into PA01?