methylation specific PCR - (Nov/14/2008 )
I'm working on epigenetic regulation in cancer methylated genes...I found many problems with MSP after bisulfite treatment.
I obtained some pairs of MSP with the respectively unmethylated pairs using MethPrimer but in any case i cannot obtain the desired band.
I'm sure that the bisulfite treatment is working because in my promoter there are two CpG islands and for one of these i can use bisulfite primers and with these i got nice results....
i have not idea how can i solve this problem, i tried to decrese the anealing temperature in the MSP PCR, increase the time of denaturation in MSP PCR, to add Qsolution (because i generally use Qiagen taq) in the mix....the last try i can do is to cross methylated and unmethylated primers, but i'm not so confident to get some bands.
I hope that someone could help me!!!! please i'm completely stuck at this point.
Show me your sequence (original, bisulfite treated) you want to analyze.
can you elaborate on your results as to what you mean you are not getting the bands you are looking for?
do you get any bands? if so are they the wrong size? are they coming up in samples that should be coming up?
This information would certainly help us all troubleshoot for you.
I obtained some bands but most of the time smaller then the expected one; this wrong bands are common to all the samples but i'm not expecting any differences between different samples.
When i'll get the bands the differences will be in the cycle sequenced samples........methylated or not methylated.
I hope that this further information will help your advice.
Answer for methylink......i posted a word document with my sequence of interest as you asked me
what is your methylated primer sequence?