PCR Primer Dimers

Troubleshooting PCR Primer Dimers

This is a PCR troubleshooting page for troubleshooting the PCR primer dimers band/product problem and its solution.

Primer Dimer Products or Bands Problem

Long Non-Specific PCR Products are usually encountered when you run your PCR reaction on an agarose gel and you visualize very small PCR bands (less than 100 bp in size). The bands would be approximately the size of your primer (if they are self-annealing) or about the size of both your primers (A and B) together (if both your primers are annealing together).

This formation product of the primers annealing together are "primer dimers" and are formed by the annealing of your primer with itself or with the other primer. The solution to primer dimers usually involves using less primer or decreasing the annealing of primers to themselves, and increasing annealing to the target sequence on the template DNA.

Primer Dimer Products or Bands Solution

• You may be using too much primer. Use less primer in your PCR reaction
• Your primers may be annealing to each other. Re-design primers 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
• Try adding DMSO up to 5%.
• Try using HotStart PCR instead of regular Taq polymerase PCR.
• Conduct PCR with and without formamide.
• Titrate Mg2+ (MgCl - 1.5, 2.0, 2.5 and 3.0 mM) concentration.
• Increase the DNA template amount (concentration).
• Increase the annealing temperature (try optimizing using a gradient PCR machine to find optimal temperature for annealing).
• Use a combination of some/all of the above.

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