Problems with Multiplex PCR - Problems with Multiplex PCR (Sep/08/2010 )
Iím trying to do a multiplex PCR using 4 different primers to get a 1kb and 4kb fragment. The melting temps of the primers are all between 54-58, therefore I;m using a 55C annealing temp and a 72C extension step and 30 cycles. When using the individual primers I get a 1Kb fragment and a ~4Kb fragment but when using all 4 primers together I only see the 1Kb fragment. I use 0.5ul out of a 10Mm concentration of each primer in a 20ul reaction, and use 2ul of DNA. Any help on trouble shooting this will be greatly appreciated. I'm at a very tight deadline so any help is much appreciated to get this experiment going Thanks!!!!..
First of all, why would you want to do such a long amplification in your multiplexes?
However I used to do this before my having multiple target varies from 1kb to 5kb in a single run untill someone advice me not to do this. Usually for multiplex PCR the longest would be less than 1kb.
Ok, maybe for some reason you have to do this way (mind to share?), I would first advice you to do a gradient PCR range from 50 to 60C. You might add DMSO or other addictive (5% DMSO works for my multiplex). Also, check your primers whether is it self binding with each other and forms primer dimers. And maybe your 1kb fragment have multiple copies and you need to greatly reduce your primers set for 1kb, and greatly increase your primers for concentration for 4kb.
Hope this helps.
If you are on a tight schedule then you should skip the multiplex pcr and do two separate pcr reactions, which are already working. You have no guarantee the multiplex reactions will work, or that they will work without changing the primers.
What extension time are you using in your multiplex PCR?
Assuming you have to multiplex (since as someone already pointed out if you do not have to it is better not to for this size amplicons), then one of the first things you should do is reduce significantly the amount of primer you are using for the 1 kb amplicon. Lower it by as much as 90% of what you are using right now.
My two cents