Tuesday, August 11, 2009

Sooo...
Yesterday's transformation of cloning vector pTOPO (carrying comM) into DH5-alpha was supposed to yield blue and white colonies, depending on whether the correct gene was inserted into the plasmid but...
I got very nice pearly white colonies (OK, not exactly pearly, but white nonetheless) for the comM and ligA vectors, but recBD didn't grow much at all, so I replated the recBD cells from yesterday (E.coli magic; you can't "replate yesterday's cells" with H.in).

There are, of course, a number of things that could have gone wrong:

1. Ligation didn't work properly.
I have the ligation products, so maybe I could run them on a gel after cutting with a restriction enzyme?

2. Transformation didn't work properly.
Try it again?

3. Plating didn't go over so well.
I've checked my calculations for ampicillin and X-gal.
Amp stock = 50mg/mL
X-gal stock = 40mg/mL
Want final amp concentration of 100ug/mL and final x-gal concentration of 80ug/mL.
So, to 50mLs LB agar, I added 100uL amp and 100 uL x-gal.
That works out...



Also transformed KW20 strain with dprA::spec construct that SS made last week. Later, I'll use this new dprA::spec strain to test the transformation frequency of the RecJ-ExoI-dprA- triple mutant.

No comments: