Multiplex PCR - (Sep/07/2011 )
I did an multiplex PCR experminent with two primersets.
I first tested both primerpairs individual, and they are very specific, but when i add them together, they also amplify other products besides the two products i designed them for.
I have to admit that the FP of the first primerpair overlaps one nucleotide of the RP of the second primerpair. Should this be an issue? So not, what could me the problem and how to solve this?
Primer dimers can be a problem, especially if they are stable. You can use software as the usual primer design programs to find out if the primers don't fit good (primer 3, perlprimer, netprimer) or this software, that is not bad.
To avoid this you may design new primers that do not form dimers and/or other structures you don't want, you can try to add additives that sometimes help (BSA, betaine, etc), and a hot-start often helps, too.
Usually, using a higher temperature will solve the problem. Try add DMSO 5%. Possible to show your gel here?
Along with Adrian's high temperature, if possible, slightly reduce the concentration of the primers you are using for the multiplex.
I didnt run them on gel, i saw the aspecific product on qPCR.
I'll try to sent the figure.
i just can not find out how to past/import my figure
try to "print screen", paste and edit in "paint", save and upload to "imageshack", finally put the link here?
here he is:
the red curve (highest one) is the aspecific product
I'm sorry, I dont think you had put the correct link...
you can also post it as attachment (hidden in more reply options) here