About MSP result ! - why always have two bands (Aug/30/2005 )
I have performed MSP to determine the methylation of a tumor suppressor gene.10 normal tissue and 10 cancer tissue were employed as PCR template,and the PCR product were visualized in 2.0% agarose gel.My problem is all the sample have two bands:methylated bands and unmethylated bands. methylated bands were weak in normal tissue ,but unmethylated bands were weak in cancer tissue.I can understand the two bands in cancer tissue.(cotaminated with normal cell),but Why there were two bands in normal tissue? HOW to solve this problem?? optimize the PCR condition or something else?
how many cycles are you performing for your PCR??
if you reduce the number of cycles you will find that the faint bands will not be visible, there will be methylation differences between cell to cell and you could say that there was "contamination" or cells in the normal population that contained the unmethylated allele.
Hi nick :
I have performed 36 cycles in MSP.Is it too much?
How many cycles in your recommendation?
36 cycles does seem a bit high, I am sure if you reduce the number of cycles, to say 30 or maybe even lower to 25, you will only see the band that was bright originally and nothing in the sample with the faint band.
with that number of cycles, I think you are saturating the reaction.