MSP trouble - (Feb/23/2006 )
I got both 'U' and 'M' bands with my MSP primers, but I am pretty sure that the control DNA was not methylated. I put the 'C'/'T' alternatives on the 3' end of the primers for 'M'/'U' amplifications. Did anybody have the same problem? Do you think the amplification will be more specific if I incease the melting tempratue?(I use 55C) . By the way, which kind of enzyme do you usually use for MSP? I use Qiagen hotstar Taq (35 cycles). Is there any enzyme better than this one? Many thanks.
Sorry, I mean the annealing Tm (not the melting Tm). If higher annealing Tm will give more specific product? Thanks.
sure can!!
but have you ruled out that you have a mixed population of cells? which will give you the bands you see.
Nick
but have you ruled out that you have a mixed population of cells? which will give you the bands you see.
Nick
I have the same promblem with Haiyan, I got both 'U' and 'M' bands with the positive control DNA ,which should not have the U band as other literatures have said,but the negative control have only one U band.I think i have no rcontamination when extracting the gDNA.
Do you think that primers designed from the Methprimer is specific?
I do the nested PCR.The first round is 40 cycles,and the second round is40 cycles,too.
Could someone help me ,please?Thank you very much!!
two rounds of 40 cycles is way too much for MSP. any background signal will come up as a false positive otherwise.
Try reducing the number of cycles to say 30, 25 or even 20 cycles. 40 cycles will saturate the PCR.
Nick