questions about degenerate primer PCR - (Nov/30/2001 )
Dear friends: I am performing the degenerate primer PCR and tried many different conditions, but my expected-size product cannot be seen and smeared bands always appear. whoever is experienced in this field gives me some valuable suggestions ?
Smeared bands usually means too much DNA is being loaded or something is wrong with the buffer in the gel or the buffer in the rig that you are running the gel in.
When using degenerate primers, I am usually successful with a Thermocycling program where I do an initail 94 degree denaturation for 2 min. Then, I do three rounds of amplification with an annealing temp of 35, followed by three rounds of amplification at 45 annealing temp, and finally about 25 rounds of amplification using a 55 degree temp.
After these 31 rounds, follow up with a 72 degree extension for 10 min.
Good luck and remember that just because primers should work in theory, doesn't mean they always will- maybe you should try getting a new set of primers.
Yep. Same as the previous reply. Start with low annealing temp for a few cycles and then increase to the expected tm of primers. Should work generally.