mutagenesis - (Aug/26/2009 )

Hi mates,

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.


should set the annealing temp to high levels to avoid the unspecific binding of the primer extents t the insert.?
Is there any problems that i will face when using such a long primers?

Best regards

thegene on Aug 26 2009, 09:52 AM said:

I've done PCR using genomic DNA as template with a primer about 18 nts of gene specific and 42 nts of a tag I added. It worked fine.

Initially, use the annealing temp of the gene-specific sequence only, otherwise it may not bind at all. Since you're working with a plasmid, you will probably have less trouble with non-specific binding than you would if you were using genomic DNA. However, there's still a possibility of it. If it ends up having a lot of nonspecific, you can either increase the annealing temp all around a little bit, or start the first few cycles with the lower temp and then raise it higher so that the primers bind more specifically to the products having already been made in the first few cycles.

I'm sure it will work, but it may not the very first time. Just need to optimize if it doesn't.