3 replies to this topic

[Boba]

Hello,
Was wondering if anyone can help me with my MSP.
I used methprimer to design primers for a bunch of genes. I used the Tm from metprimer which ranged from 55-59 degrees. To use the same PCR I used an annealing temp of 50 for 40 cycles.

I am getting single bands at the right size but my U primers and M are picking are not discriminating between unmethylated and methylated DNA.
I used Qiagen control M and U DNA and some cell lines. My unmodified DNA is not being amplified and primers to unconverted DNA are picking up bisulfite converted DNA. I am using EZ DNA methylation kit and treating 500 ng.

What could be going on? Could annealing temp have anything to do with this?
Thanks!

Edited by Boba, 20 April 2010 - 07:40 PM.

[pcrman]

Try raising the annealing temperature to 55C.

Regarding U and M primer indiscrimination, this is a common problem with MSP method. PCR conditions need to be optimized before results can be trusted.

If your regular PCR primers are picking up converted DNA after raising annealing temperature, probably the primers do not contain enough non-cpg Cs especially at their 3' end, or the DNA is not completely converted.

[Boba]

Thank you. I'll try that. By the way, do you have preferred DNA reagents for amplifying bisulfite-converted DNA?

pcrman, on Apr 21 2010, 10:19 AM, said:

Try raising the annealing temperature to 55C.

Regarding U and M primer indiscrimination, this is a common problem with MSP method. PCR conditions need to be optimized before results can be trusted.

If your regular PCR primers are picking up converted DNA after raising annealing temperature, probably the primers do not contain enough non-cpg Cs especially at their 3' end, or the DNA is not completely converted.

[pcrman]

Yes, I recommend that you use JumpStart Red Taq from Sigma for the PCR.