2 rounds PCR got problem - (Mar/03/2011 )
I am trying to insert restriction sites and a HA tag into my target DNA fragment by a 2 rounds PCR.
I always got a good product after 1st round of PCR and used it as the template for the 2nd round but could never get good result.
Would it be the problem with my primers? Because I used same reverse primer for both 2 rounds of PCR.
When I need to run 2 rounds of PCR to create the DNA fragment I need for cloning, I try to make sure there are extra base pairs at both ends of the first amplicon for the second set of primers. In other words, think nested PCR: the second set of primers should be well within the ends of the first amplicon. The rationale is that primers anneal better when they are within a DNA fragment and much less so when they need to anneal at the end of a DNA fragment.
ivanbio on Thu Mar 3 17:37:00 2011 said:
When I need to run 2 rounds of PCR to create the DNA fragment I need for cloning, I try to make sure there are extra base pairs at both ends of the first amplicon for the second set of primers. In other words, think nested PCR: the second set of primers should be well within the ends of the first amplicon. The rationale is that primers anneal better when they are within a DNA fragment and much less so when they need to anneal at the end of a DNA fragment.
Thank you. I designed an extra 'GCG' at both ends. Is it enough? I cannot put too many extra base pairs because I already had some extra uncomplimentary base pairs to introduce my restriction sites and tag.