cloning two fragments with deletion in between - PCR primer design (May/15/2006 )
I want to create a vector insert, by creating a deletion between two fragments of a gene.
To do this, I was advised to PCR these two fragments seperately, whilst introducing sequence homologous to the other fragment, at their desired joining ends, by primer design. A second PCR with the new templates, now slightly complementary, will give me one long insert with a seamless joint.
My question is on the primer design on the first PCR: as I need to introduce enough 'alien' bases to the 5' of my primers, so that there is enough complementarity in the second PCR, what should the primer length be? Perhaps 15 'alien' bases followed by 20 template-specific bases? This should then give me 20 bases for a good PCR in the first round, and 15x2 bases of complementary sequence for joining in the second round. Any suggestion/comment?
Thanks in advance. (My first ever post in anything)
15 to 20 bases should be fine. Perhaps. You'll just have to try it and see.
Thank you. I will try 21 bp specific bases plus 15 non-specific ones for annealing.
for all the mutants that i have created through pcr based site-directed mutagenesis, i have used primers approx 25 bp long, keeping the tm between 45-50 c. it is the overlap in the sequence that would qualify the site of mutation that is important, and should be adequate.