Friday, July 17, 2009

Long live PCR!!!
Lane 1: 1kb DNA ladder
Lanes 2-4: SS's KW20 DNA with RecJ, ExoI, and Hi crp primers, in that order
Lanes 5-7: my KW20 DNA with RecJ, ExoI, and Hi crp primers, also in that order
I ran this gel for 45 minutes at 80V.

The bands are quite gloppy. Is it the new Taq?

The RecJ gene (HI_1214) is 1728 bp long, and the ExoI gene (HI_1377) is 1422 bp long. Both are interrupted by a tetracycline-resistance cassette (how long is it?). Hi crp is 675 bp long, so it's much smaller than what we expect for RecJ and ExoI.

I think the second band in the ExoI samples is due to annealing temperature (which was 45 degrees C for this reaction). SS also got a smaller second band in one of her PCRs, and she was going to lower the annealing temperature. The Tm (I forgot what the "m" stands for, but I think it means the temperature at which half of the primer is attached to the template DNA) of my ExoI_F primer is 57.2 degrees C, whereas my other primers (RecJ_F, RecJ_R, and ExoI_R) have Tms in the 47-50 range. So I guess the low annealing temperature made the ExoI_F primer bind with parts of the template that it was not specific to.

Happy weekend!

No comments: