I am working with a doubly methylated cytosine base which is at 12th position in 30 mer oligonucleotide. The forward primer is cy3 labeled. I am trying to hybridize it using opposite pairing of A, C, T, G but there are problems in hybridization. I used 1:1 ratio of forward and reverse oligo in STE buffer and heated it at 940C for 4 mins and allowed it to cool down to 230C for 2 hours. But The oligo is not hybridized well and there is, even more, a problem in hybridization where there is a mismatch like C:C C:A. How could I solve this problem? Thanks
use a longer primer to stabilize the duplex even with a mismatch, which is destabilizing.
Hi there,
In theory a single mismatch in a 30pb fragment shouldn't be a problem. I would suggest a higher temperature for denaturation (98°C for at least 5 minutes) then go for a very slow cooling step: ideally using a PCR block to control temperature going from 98 to 20°C in a. few hours. Alternatively, boil the sample in a large volume of water and then let the waterbath cool down to 20°C with the samples in it.
I'd be tempted to use one of the degenerate or wobble bases available from a variety of companies. They can form non traditional pairs and have seen some use. Inosine is a classic example but I believe there are many better options around now!
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