I have designed primers to give me products of 100-140 base pairs, with the intention of real-time RT-PCR in the future. After a normal RT-PCR run I have noticed moderately strong primer dimers in my agarose gels. I have read that primers dimers could really be a problem in the real-time application.
How can I prevent/minimise the formation of primer dimers?
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1 reply to this topic
Posted 03 August 2003 - 02:28 PM
sometimes you can optimise the concentration of primers you use for real time, and this can reduce primer dimers (ie. less primers). the other thing to keep in mind is that you can determine from a melt curve of your products where to read your dye. you can set the read temp above where the rpimer dimers have melted, but below where your product melts (the melt curve is generated by the real time machine). however sometimes you just cant avoid some primer dimers, and if they continue to be a problem, you may need to design new primers