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Hi all,
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!!
methylmouse
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Mysterious peaks in pyrosequencing runs!?
06 August 2012 - 04:07 AM
Hi all!
Our lab recently started to use pyrosequencing for quantitative methylation analysis. We all do not have a lot experience and expertise with this technique.
I am currently investigating the methylation status of one gene in tumor tissue samples. I have sequenced more than hundred samples and for a few samples I made the following observation: The pyrogram shows peaks at dispensations, where no peaks should occur. When I analyze the corresponding PCR products by gel electroforesis, I see a nice band at the good size, plus a slight band below the good band. It looks like a dimer, but why do I only see this in combination with the peaks? Could this also be contamination?
I always include a negative control (no DNA in PCR mix) in the pyrosequencing runs, which do not give any results (= clean). I checked my primers with MethylBlast and they should be specific.
I would be more than happy, if anyone could help me looking for a reason for those peaks/bands!! Did someone make similar observations?
I cannot analyze my data until I know, what I am dealing with...
Cheers
methylmouse
Our lab recently started to use pyrosequencing for quantitative methylation analysis. We all do not have a lot experience and expertise with this technique.
I am currently investigating the methylation status of one gene in tumor tissue samples. I have sequenced more than hundred samples and for a few samples I made the following observation: The pyrogram shows peaks at dispensations, where no peaks should occur. When I analyze the corresponding PCR products by gel electroforesis, I see a nice band at the good size, plus a slight band below the good band. It looks like a dimer, but why do I only see this in combination with the peaks? Could this also be contamination?
I always include a negative control (no DNA in PCR mix) in the pyrosequencing runs, which do not give any results (= clean). I checked my primers with MethylBlast and they should be specific.
I would be more than happy, if anyone could help me looking for a reason for those peaks/bands!! Did someone make similar observations?
I cannot analyze my data until I know, what I am dealing with...
Cheers
methylmouse
