touchdown qPCR - using degenerative primers (Dec/03/2007 )
I am wishing to do qPCR on a complicated region looking for copy number variation. The PCR primers that I have designed are degenerative and when i do a normal PCR reaction i get lots of bands. I am thinking about doing a touchdown PCR
BUT dont know if then it would be useful for qPCR?
thanks for any advice
you could always do a test run on a few samples, just to see if you like the result.
If you are having multiple bands, there are always other options to try (magnesium, DMSO, etc). We had a rep tell us that sometimes changing the amount of each individual primer can help. The reasoning was that each primer binds differently, with different efficiencies, and so changing the proportion of each can help the reaction.
you could two a two step PCR where the first step is to enrich for your template using touchdown for a very limited number of cycles (to ensure all your reactions remain in the linear range of amplification) and then using the product of that PCR as template for your qPCR...