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 DH5&alpha;

Summary:

I transformed the pKn005.L3 and L4 ligation products into DH5&alpha;. 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.