problem in cloning PCR primer design with restriction site - (Mar/12/2013 )

Dear friends
Im designing a primer pair for amplification and cloning and expression in pET expression plasmid. I have chosen Nde1 and Xho1 restriction sites for the purpose.First i have taken the gene to be cloned and designed a primer pair as described below
1. Forward primer - length 22 bp, Tm - 54, GC - 27 %
2. Reverse primer - length 22 bp ,Tm - 52 GC - 24 %

After adding the restriction site and 6 additional bases (to both primer sets) Tm increases to 74, GC % of the primer set is 35 %

Im unable to get good primer pair

Please help

I think you are doing fine. the new parameters for the primers look fine, as long as the two primers do not have a big difference in Tm. Another thing is you don't necessarily need to add 6 nt to the end of the primers, some restriction enzymes require less extra bases. You can take a look here: https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

You have a very low GC organism. I would be using a longer binding region for your primer, perhaps 26 or more bp. The Tm of the primer with the extension is relatively unimportant -- you should concentrate on the Tm of the region binding to your template.

With your low GC, you should also consider lowering your extension (not the annealing) temperature, perhaps to 64 rather than 72, and doubling the extension time. This will allow you to PCR very low GC regions, which otherwise cannot be extended.

pcrman on Tue Mar 12 19:19:49 2013 said:

Essentially yes. Some enzymes require more (up to 6).