Pcr cloning - how to get a single band (Jun/16/2002 )
Hi, there. I met some problems in the pcr. Actually what I did is degenerative Pcr. When I tried, I always get multiple bands. If I increase the temperature, then just no bands left. So, should I modify the cycle number, the annealling time, or the extending time.
how about hot start
what I used is hot start
maybe you can decrease the annealing temperature for the 10 first cycles and then use a higher for the additionals...this can decrease non specific amplification especially in the case of degenerative PCR.
I've experienced this problem in the past. I'm afraid it's the nature of the beast. Your degenerate primers consist of many different sequences which may bind to many places in the genome or library with which you are working. You can try variable annealing temps which has been suggested. You may vary the Mg++ conc. Do you have any idea how large the amp. product should be? You may need to clone all the bands and sequence them. If there are only a few this may be a good strategy.