PCR anealing temperature question - (May/07/2006 )

Is there additional sequence in your primer besides the initial annealing sequence? For example do you have a restriction sites designed in the sequence or linkers, etc? Calculate the TM for the compliment to the initial template and try that temp. It may be below 58.

I'd also trouble shoot the PCR reactions.

The length of both primers are 30 bps, which include 20 bps initial template sequence and 10 for restriction enzyme and starting codon. The %GC contents are 53.3% for 5' and 60% for 3'. The Tms decrease to 65-70 based on different formulas you have suggested. The Tms are remain same if I taking into account salt concentration.

I think the temperature may be not the only problem, since I found there are 5 consecutive bases pair to each other between 5' and 3' primers. I think that may form primer dimers and decrease the yield of pcr product. Do you think I can get rid of all possible primer dimers if I increase anealing temperature all along or adding DMSO? (I tried, but I didn't see big difference)
Thank you

Yue