Hi, I am trying a PCR reaction that is giving me a bit of trouble. My template is genomic DNA from yeast (s.cerevisiae) and at this point I want to check the quality of the gDNA preps. I have loaded aprox. 1 microgram on a 1% agarose gel and I got a pattern with two bands, 1 above 10KB and the other close to 6KB. If i boost the signal i can see even a really faint around 3 KB band. Is this a normal pattern?? what should I expect? the bands are normal, no smearing
it would be better to see a picture but in general I am not very worried about some degardation of dna. RNA can be a problem in that if OD260 is used to calculate amounts then you may end up using not enough DNA but degraded DNA ( and yours sounds fine to me) is usually larger than the pcr product that you are trying to amplify so I would expect this DNA to work fine as a pcr template
The normal way to check the prepared gDNA is to run a low percentage agarose gel and measure the OD260/280. The purified gDNA should be no or less smear with very large length on the top near the loading wells with a OD260/280 ratio roughly of 2.0. Since the the template gDNA is very big, whatever primers you chosen for your PCR amplification, there are always chances that your primers matched onto different regions, which give you different PCR products although you might get the only expected products if you play with the annealing temperature carefully.
- Badges
- Science method