multiplex PCR - (Dec/07/2003 )
HI, friends, I have two pairs of primers with totally different Tm. I tried multiplex PCR in one tube and with one cycling conditions, and only one pair of primers worked. I wonder whether I can still put those 2 pairs of primers in one tube but run with one cycling condition first for one product and then go on to another cycling condition for another product. Thanks.
I don't know what you exactly meant by cycling conditions: Ta or cycles? If there is a difference in Ta, you can select a Ta half way between those two Tas, since Ta usually works in a wide range. Or you can redesign a pair of primers with Ta close to the other pair. If there is a difference in cycling number, you can adjust the amount of primers added to your PCR reaction. For example, if you amplify the two genes using 30 cycles, and only one show product, then you should reduce the amount of primers for that gene and increase cycles.
You need optimization (means repeat and repeat) before you can get a desired picture. Also there are software which can design multiplex primer (search google).
Hope it's useful.