Assembly PCR - Design of PCR program for primers w/low Tm? (Aug/13/2007 )
First post, but I visit often as I'm a 1st year grad student and find this site quite useful as a reference.
My question is on Assembly PCR. For those not familiar with it, assembly PCR uses a set of overlapping/complimentary primers (40mers in my case) to assemble genes. The problem is the gene I need is quite short (120nts) so the overlap and hence Tm of all of my primers is ~52C +/- 2C.
Each primer has ~12 nt overlapp to its complimentary primer(s).
So to the question: With Tm's so low, what should I set my annealing temp to in the PCR temp gradient? I'm thinking as low as 40C because I'm not really worried about getting non-specific products since there's so little material to begin with. Thanks.
-Mike
My question is on Assembly PCR. For those not familiar with it, assembly PCR uses a set of overlapping/complimentary primers (40mers in my case) to assemble genes. The problem is the gene I need is quite short (120nts) so the overlap and hence Tm of all of my primers is ~52C +/- 2C.
Each primer has ~12 nt overlapp to its complimentary primer(s).
So to the question: With Tm's so low, what should I set my annealing temp to in the PCR temp gradient? I'm thinking as low as 40C because I'm not really worried about getting non-specific products since there's so little material to begin with. Thanks.
-Mike
Is it possible to increase the gc content of your primers in order raise the Tm of your primers?
Why not just have a slightly longer overlap? Even up to 20mer overlap is OK, and synthesising a 50 mer is no problem nowadays. Or you could just get two 70mers with 20bp overlap, and two regular primers for the termini. I know the total yield of the 70mers won't be fantastic (maybe 30-50%), but I have made primers that were 95mers, and still managed to make the construct.
OK, make the 70mers. Mix them and do 5 rounds of amplification. Take 1ul of the product, and do a regular PCR using the two terminal primers. Voila, one 120 bp construct.
Good luck.