Problems with Multiplex PCR
Posted 08 September 2010 - 01:40 PM
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!!!!..
Posted 08 September 2010 - 04:52 PM
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.
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Posted 08 September 2010 - 05:50 PM
Posted 09 September 2010 - 03:37 AM
Posted 09 September 2010 - 07:23 AM
My two cents