How can I increase the efficiency of multiplex PCR with more than 3 pairs of primers? Primers do not form stable dimers and hairpins (less than 5 nucleotides) and work in singleplex PCR quite well. But when I try to do multiplex PCR with 4-6 pairs of primers the efficiency of PCR (conventional format) greatly reduced.
Hi Dmitry,
You should check the concentration of dNTPs you used in the multiplex PCR. Probably you need to increase it with the increasing number of oligos and amplifying targets.
when you increase the primer or ntp concentration you do 2 things...you take more Mg out of solution and you change the Tm of the primers slightly so I would add a little more Mg and lower the annealing temperature also. It may be worth putting 1M betaine ( final concentration )in the mix as this makes the dna template melt easier and generally improves the efficiency of the pcr. I would leave the ntp concentration alone as they are in huge excess already ( as are the primers usually)
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