PCR with a very long and a short primer
Posted 14 November 2011 - 08:23 AM
I am trying to insert an epitope tag at the end of my protein and I want to use PCR to clone the fragment in. Now the problem is the reverse primer is very longer as it incorporates the tag (nearly 100 nucleotides) in comparison to forward primer (20 nucleotides). For the first round the reverse primer anneals to the 20 odd nucleotides of the template DNA while the tag sequence is in the tail of the PCR, however for the subsequent rounds the reverse primer now is as long as 100 nucleotides which is no match for a 20bp forward primer.
Has anyone encountered a similar problem. Any information would be useful
Posted 14 November 2011 - 09:46 AM
I always had an alternate hypothesis....
Posted 14 November 2011 - 01:43 PM
Posted 14 November 2011 - 02:50 PM
Posted 30 November 2011 - 10:09 AM
Posted 01 December 2011 - 12:28 AM
Maybe I would try to play with DMSO.
I never trust anything that can't be doubted.