Wednesday, July 22, 2009

I ran a gel on yesterday's PCR products, which were MAP7, RecJ-, ExoI-, and RecJ-ExoI- DNAs from the Oxford lab with ExoI primers.
The MAP7 and RecJ- lanes are aligned with the 2kbp rung. This shows that the ExoI gene in this samples is uninterrupted. The ExoI- lane is a little higher than the RecJ-ExoI- lane because the ExoI gene in the former is interrupted by a tetracycline resistance cassette, whereas the ExoI gene in the latter is interrupted by a kanamycin resistance cassette. The ladder's not too clear, but I'd estimate that the tet cassette is ~3kbp long and the kan cassette is ~1kbp long.

Well, now we know what the RecJ-, ExoI-, and RecJ-ExoI- DNAs are supposed to look like with RecJ and ExoI primers.

Today I also extracted DNAs from my RecJ-, ExoI-, and RecJ-ExoI- strains. I'll be running PCR on these to check that they look like the Oxford DNAs. The kit was a lot faster and easier to use than the manual extraction method. No phenol-chloroform, too.

Lastly, I counted colonies from a transformation I did yesterday with KW20, RecJ-, dprA-, and RecJ-dprA- strains and MAP7 DNA. The cells from this experiment are a different batch from my previous transformations, so I got a good biological replicate. This set also showed RecJ-dprA- cells are not great transformers.

No comments: