Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 8.11.08

8.12.08

I doubt I'll have a chance to transform the Lox/Gentamicin cassette into Pseudomonas, but I can at least try to finish generating the cassette for HmgA.

To Do:

• Submit pKn006 preps for sequencing.
• Try cloning the cassette into pKn005 using both sites.
  • Digest pKn003.P1 both with BamHI and XbaI (in separate reactions). Blunt end both.
  • Digest more pCM351 with NdeI and SacI. Run out on a gel, purify, and blunt end.
  • Ligate both into each other using a high ratio of cassette:vector.
• Transform ligation products into DH5α

Summary:

I transformed the pKn005.L3 and L4 ligation products into DH5α. Hopefully will get colonies tomorrow.

8.13.08

As of 0800 this morning, there are no colonies on any of the plates from yesterday's transformation. Looks like this cloning step just wants to be difficult. There's not really much else I can do here.

Before I leave today, submit sequencing for both pKn006 clones.