I used the methylation specific PCR technique to detect the percentage of specific gene DNA methylation by comparing with unmethylation band. I try the condition to resolve the non specific band in the unmethylation condition ( Increased Tm of annealing) but I still found the non specific band (the band size is more than specific band). In addition, when I increased the annealing Tm more than 2 degree Celsius, I cannot detect any band. How do I do to remove the non specific band?
Thank very much
Hi Wiyada,
Add additional primer sequence (e.g. M13 primer sequence) to the 5 prime end of your forward or reverse methylation or unmethylation specific primers. This additional sequence contain all four bases and provides universal sequencing primer binding sites. This also helps in increasing the melting temperature of the product and specificity.
For detailed reference and protocol, I have attached the handbook from Invitrogen
Hi,
You should check your bisulfite conversion method as you are getting non specific band in unmethylated set of PCR reaction which is based on bisulfite conversion of unmethylated cytosine nucleotide.
Actually, in my case, I will try to design other unmathylated primers. It is usual and difficult to design the unmathylated primers. Try several ones, and you will succeed.
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