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Amplification with U primers, but not with M primers


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#1 ClaudiaN

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Posted 12 March 2013 - 07:05 AM

Hi,

I've got no experiences in this field of research. I want to analyze the methylation status of the murine Ppar gamma promotor in cells under control conditions vs treated cells. I'm expecting promotor methylation after treatment. I designed primers for MSP with MethPrimer software. 4 CpG islands were found and I designed primers for the biggest one. TM for the primers is 59-64°C. I did PCR with a annealing temperature of 55°C, 60°C and 65°C and with HotStar Taq Master Mix. I always got specific PCR products with the U primers, but never with the M primers.

Can anybody help me?

#2 pcrman

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Posted 12 March 2013 - 11:25 AM

Your result may indicate that there is no methylation. To confirm that, you have to first prove that your M primers work. How to do that? You can use positive control DNA (full methylated - see this post: how to create fully methylated DNA) or DNA from cell lines in which the promoter is known to be methylated as controls. Alternatively, you can verify your MSP results by performing BSP--designing BSP primers to amplify the same region and sequencing the product.




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