manual primer desgining - (Jul/28/2008 )
I am trying to design primers manually,unfortunately reverse primer GC percentage is not reaching more than 42, forward is coming upto 50 or even more,how much GC percent reverse primer should have,is it necessary to have GC percent more than 50 for two primers to isolate ORF fragment.can any one help in this issue.
thanks in advance,
It's nice to have a good GC content, but not really necessary. I work with low-GC organisms and often can never get up to 50% GC. You'll just need to make your reverse primer a bit longer so the Tms of forward and reverse are nearly equal.
I agree with wbla. Just aim for the same tm for both forward and reverse primers. Since you are building your primers manually, Northwestern oligo calculator is very useful to check for both the tm and presence of hairpin loop structure and self complementation. Try to minimise the hairpin loops and self complementation.
I personally try to aim for a higher tm of around 57 Celsius to 60 Celsius. Higher tm, make the reason more specific and help break the self complementation when it does arise.