Monday, July 20, 2009

I started off the day with some PCR. I ran RecJ primers with KW20, RecJ-, ExoI-, RecJ-ExoI- DNAs (the last three are from the Oxford lab). I did this experiment as a control. The results will show what we expect from the genotypes from the Oxford lab. Eventually I'll run PCR with the same types of DNA, but extracted from cells that I've transformed using the Oxford DNA.

It basically looks like this:

1. Oxford DNA => test with PCR
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2. I use it to transform wildtype cells into RecJ-, ExoI-, and RecJ-ExoI- cells
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3. I extract chromosomal DNA from these cells => test with PCR

I want to check that the DNA from step 3 is the same as in step 1.
The gel came out as expected. KW20 and ExoI- bands are lower because they're shorter; they're not interrupted by a tetracycline resistance cassette. RecJ- and RecJ-ExoI- bands are higher up on the gel because they're longer. The gene coding for RecJ has been interrupted by a tetracycline resistance cassette. They're quite faint, though. I suspect elongation time needs to be increased. So the gene for RecJ is about 2kbp long, and the tet cassette is about 2 or 3 kbp long (I'll have to check that somewhere).

1 comment:

Sunita Sinha said...

Good work Rhena! Now lets try the exoI PCRs too.