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primers for cloning - (Jul/29/2014 )



I've designed a pair of primers (with restriction sites) to clone my gene of interest. How do I determine the annealing temperature for PCR?  I have used some online software and they show:


forward primer:

GC content: 64%

basic Tm=75 deg

salt adjusted Tm=70.


reverse primer:

GC content: 46%

basic tm= 67 deg

salt adjusted tm=62 deg


many thanks



First, understand that these numbers are approximate, and aren't something  to pay a lot of attention to. Your PCR will either work or not, and if your primers are sufficiently specific, the melting temperature is unimportant. You should calculate based on the portion of the primer which binds the template during the first few cycles (that is, ignore the restriction site and any extensions). Aim for a 20-26 bp matching region with about 50% GC and a 3' G or C. When you try your PCR, anneal at 55, which almost always works. When it doesn't, try a gradient from 48-65. If your primers still don't work, then redesign them. If you have very high GC content in your template, you could try high GC buffers, or addition of 4-6% of a 5M betaine solution. If you have very low GC content, then you could try lowering the extension temperature (not the annealing temperature) to 65 from 72, and lengthening your extension time.