No PCR products in MSP with MethPrimer designed primers - (Sep/22/2005 )

Hi! my friends here. I am new. Recently, I have tried MSP on 10 tumor supressor gene (TSG) using Chemicon CpGenome DNA Modification Kit and MethPrimer designed primers (M and U). It turned out:
1. #3 is partially methyalted because both primers M and U have PCR products.
2. #6 is not methylated because only primer U gives a band.
3. #9 is methylated because only primer M gives a band.
4. All others (7) have no band with either primer M or primer U. Why? This is my question.

The conditions: 35 cycles, annealling temperatures were given by Methprimer. They are between 56-60C. I did separate PCR for different annealling tem. The 7 TSG had no band. Then I tried 53C for all of them in one PCR. Still got nothing.

Can some friends help me analyze the results?

And an additional quetion is how to distinguish true M and U amplifications from mismached amplifications because usually annealling tem is lower than Tm in MSP.

Thanks a lot.

Mickeymeth

the 7 primers that did not work may well be not optimal for MSP to the genes you want to look at.

You may want to try alturnative primers that methprimer would have selected for MSP for the other 7. 35 cycles of PCR could be saturating and it's possible to both M and U primers will amplify after this number of cycles so you may also want to lower the number of cycles as well. It's a fiddly technique I have to say!

Nick

Nick,
Thanks a lot for your ideas. I'll fiddle more primer pairs of MethPrimer.
Mikeymeth