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designing MSP primers (dimers NOOOOOOOO!)


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#1 SStamis

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Posted 04 February 2011 - 01:56 PM

designing MSP for a USF site in my gene of interest. These primers are going to be the death of me. I'm using MethPrimer to design them, but the primer dimer problem is endless, but With such a high content of A's and T's I'm drowning. Is there ever a point where you just deal with primer dimers? I saw some posts about formamide but wasn't sure if it applied. Obviously I'm a novice.

I don't know which parameters can be tweaked to what extent. GC content, annealing temp, length, product size, etc. any suggestions or lessons you've learned?

#2 pcrman

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Posted 15 February 2011 - 07:48 PM

Hi SStamis, don't get frustrated. Primer dimmer is a big issue for MSP or any bisulfite based PCR because the low sequence complexity of the modified DNA. You can design and try several sets of primers that cover the same region, and optimize your PCR conditions. If you have not used hot-start polymerase, you should definitely try it. I highly recommend the JumpStart RedTaq from Sigma. Posting the PCR conditions you are using may help us troubleshoot your problem. Have you included any positive control DNA (modified DNA known to be amplifiable)?




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