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methylation specific PCR assay (PWS/A syndrome)

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#1 b612



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Posted 25 October 2010 - 04:41 AM

Hi everybody!!!
I have a big problem!!!! I want to do diagnostic of Prader-Willi/Angelman Syndrome (uniparenteral disomy). I need to know the methylation status. For this I use BISULFITE KIT (Qiagen). Then I have quantified DNA concentration of my samples. Then I have to do methylation specific PCR assay. I have next reactives for each sample:

Buffer 10x= 3 microL
MgCl2 25Mm=2.4microL
Taq Gold 5U/microL=0.6microL.
dNTP ´s 2.5Mm=2.4microL
Primers (maternal primer, paternal primer) (100microM each)= 1.5microL
DNA 30ng/microL.=1.5microL
Total volumen= 30microL.

I use program of PCR from a Kubota´s article “METHYLATION-SPECIFIC PCR SIMPLIFIES IMPRINTING ANALISYS”: 95ºC, 10´, 35 cycles: 94ºC 30”, 62ºC 30”, 72ºC 30”, and final extension 72ºC 10´.

Then I have separated PCR products in 3% agarose gel.

In this moment I have got amplificated only in samples treated with maternal primer.

Somebody knows Where is the problem!!???!!!

Thank you in advance!

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