PCR with long primers
Posted 26 August 2009 - 01:19 AM
I have a vector that contains an insert. What i want to do is to make mutagenesis . for that i have designed a sense and antisense primers that is around 52 nucleotides( 15 nucleotides are insert sequence specific and a 35 nucleotides contain a sequence of a binding sites for a bacteriophage coat protein that i want to add to my insert. do any body know or have any idea about the some hints or tips to do such a PCR with a long primers.
Posted 26 August 2009 - 12:28 PM
Posted 26 August 2009 - 04:17 PM
Posted 27 August 2009 - 06:55 AM
The Tm is calculated off the specific sequence, not the whole primer. Use a proofreading polymerase.
Thanks mates for all your help