PCR Troubleshooting

PCR Problems and Solutions, PCR Help

What is your PCR problem?:

Long Non-specific PCR products?

Primer Dimer formation?

Problem:

Long Non-Specific Products in PCR

When running an agarose gel you spot larger non-specific PCR bands that are not the right size.

Solutions to Long Non-specific Products in PCR:

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.

Use 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)

Combination of some/all of the above.

Problem:

PCR Primer Dimers

When running an agarose gel, you spot some very small PCR bands. These are the size of your primer or about the size of both your primers (A and B) together. These are termed "primer dimers" and are formed by the annealing of your primer with itself or with the other primer.

Solutions to primer dimers in PCR:

Use less primer.

Re-design primer and order a new batch. Make sure you use primer design software and check for self-annealing and that the primers do not share a large percentage of complementary sequence. Check primers carefully for homo-dimer and hetero-dimer formation with OligoAnalyzer

Conduct PCR with and without formamide.

Titrate Mg2+ (MgCl - 1.5, 2.0, 2.5 and 3.0 mM) concentration.

Increase DNA template amount (concentration).

Increase annealing temperature (try optimizing using a gradient PCR machine to find optimal temperature for annealing).

Try adding DMSO up to 5%.

Try using HotStart PCR instead of regular Taq polymerase PCR.

Combination of some/all of the above.