I am using Qiagen EpiTect Bisulfite Kit for detect methylation status.
I isolated DNA, quantified it, and I did PCR assay.
I have only amplification in PCR with primers for DNA methylated.
I have not amplification in PCR with primers for DNA no methylated.
I think the problem is in bisulfite treatment.
Where is the problem???
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 Water=18.6microL 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!!???!!!, PLEASE!!!