Unique unspecific product using MSP - (Jun/01/2006 )
Hi! I am using MSP primers to ampify a 250bp fragment. I am running the the PCR on templates derived from 9 different cell lines, all under the same conditions. All the 'methylated' primers work really well, and in 8 out of 9 cases, the 'unmethylated' ones also produce the product of the expected size. However, in on cell line that comes up with a rather prominent band in the methylated-primer set, there is also a band in the unmethylated lane, running at about 500bp. In this set, i can't see anything at 250bp. Not quite sure what to make out of it - any suggestions welcome!
how many cycles are you performing?
Hi Nick! I was running 40 cycles - the full programme using Roche's 'FastStart Taq' is:
1) 95C, 5min
2) 95C, 30sec
3) annealing temp, 30sec
4) 72C, 60sec
5) cycle to step 2 another 39 times
6) 72C, 10min
7) 10C hold
I have the pic attached if that's of help. My first idea was formation of a heteroduplex, but I think it's a bit odd that I can only see that happening in one of the cell lines, and only for the unemthylated primer pair...
That's interesting! I am surprised to see that in your M set after 40 cycles there is nothing coming up.
Performing so many cycles should be done so with care as the PCR can potentially pick up a subpopulation in your sample. ie: if 10% of cells within your sample was methylated while the other 90% was not, it would be picked up after 40 cycles.
I may suggest you reduce the number of cycles to 15-25 as you would more likley see the more abundant templates within your sample and not the minority. The more abundant template will come up sooner than the less.