I am currently trying to optimalize primers for direct MSP. I started with a temperature gradient (58C - 64C) and 35 cycles. However, I did not see any bands, only primer dimers... The amplicon size is small (~80 bp), so I checked the PCR products again on a higher percentage gel, for the dimers might cover the actual bands.
Running a 2,5 % gel for 45 min yielded in very very slight bands and very very thick and dark primer dimers.
To be sure, that I will obtain amplification with these primers and to see if it is worth it to continue optimalizing those primers, I performed nested MSP. A gradient with nested MSP and (un)methylated controls worked well.
For I am going to use fresh-frozen tissue material, I would like to get my primers working also for direct MSP!
What can I try to get my primers working? Can I change the number of cycles with such a short amplicon size?
Looking forward to your ideas/experience/expertise!!
methylmouseMember Since 06 Aug 2012
Offline Last Active Aug 14 2012 07:19 AM
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Epigenetics, Oncology, Developmental Biology