Troubleshooting for PCR and multiplex PCR

Troubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles.

* The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM MgCl2).

QUESTIONS and SOLUTIONS

1. I get (many) longer unspecific products. What can I do?

Decrease annealing time　　Increase annealing temperature　　Decrease extension time　　Decrease extension temperature to 62-68º C　　Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.　　Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.　　Take less primer　　Take less DNA template　　Take less Taq polymerase　　If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)　　Combine some/all of the above

2. I get (many) shorter unspecific products. What can I do?

Increase annealing temperature　　Increase annealing time　　Increase extension time　　Increase extension temperature to 74-78º C　　Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM　　Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant　　Take less primer　　Take less DNA template　　Take less Taq polymerase　　If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)　　Combine some/all of the above