Posted 22 January 2010 - 10:29 AM
I am trying to amplify and stitch two exons together in one PCR reaction using genomic DNA as the template. To do this I have appended to two of the primers a complementary overhanging sequence. If I amplify both exons in separate reactions, then mix them and reamplify using only outside F/R primers, I can get them to link together, but when I try to do the linking in one PCR it doesn't work. Also, using a cDNA template I can get exon linking in one step, but with genomic DNA I can't get it to work. Has anyone done this before? Does anyone have any suggestions? Are there additives that I should try? Or thermo cycling tips? Anything would be great.