stiching/linking/sewing pcr - (Jan/22/2010 )
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.
I haven't done this from genomic DNA but this could work ... start with the annealing temperature low (not too low) to ensure some production of amplicons then every 5th cycle, raise the temperature in increments so that the specificity is increased. It is possible that in the genomic DNA the spacing is too far to allow the amplicons to come together. You will need to gel purify to ensure you clone the product of interest.
Hope this helps,
Also ... increase the time for amplification to ensure products get made and can come together :-)
AussieUSA on Jan 29 2010, 03:18 PM said:
Thanks I'll give it a try and let you know!