Hi all,
i've bought a dna fully methylated and fully unmethylated from zymo to have a methylation control (that my primers are able to discriminate correctly and quantitatively epialleles post bisulfite treatment ) in a gene methylation pattern study. The problem is that the primer that i've tested, designed to amplify certain CpG dinucleotide, amplify in pcr only fully methylated dna, neither unmethylated nor a mixture of 50% umethylated and 50% fully methylated dna. I have excluded that the problem is dna, because using primers provided specifically from zymo they amplify all samples.
Furthermore in the mixture is present also a 50% of FM dna, so i should see something after pcr.
Does anyone have any idea why this happened? What can result in a failure of UM dna amplification ? It has some special characteristics?
Thank you!
i've bought a dna fully methylated and fully unmethylated from zymo to have a methylation control (that my primers are able to discriminate correctly and quantitatively epialleles post bisulfite treatment ) in a gene methylation pattern study. The problem is that the primer that i've tested, designed to amplify certain CpG dinucleotide, amplify in pcr only fully methylated dna, neither unmethylated nor a mixture of 50% umethylated and 50% fully methylated dna. I have excluded that the problem is dna, because using primers provided specifically from zymo they amplify all samples.
Furthermore in the mixture is present also a 50% of FM dna, so i should see something after pcr.
Does anyone have any idea why this happened? What can result in a failure of UM dna amplification ? It has some special characteristics?
Thank you!