AAT Bioquest

What do I do if my PCR isn't working?

Posted August 14, 2023


If your PCR isn’t working, the first thing you’ll need to do is troubleshoot to identify the problem. Before you start troubleshooting, make sure of two things - you’ve added all the components necessary for PCR to work and you’ve programmed in the correct cycle conditions recommended for your PCR reaction. These are the two very common mistakes that can cause your reaction to fail. Even one missing component or a slight deviation in programming the machine will result in a failed PCR reaction. 

These are some of the more common reasons why your PCR may not be working and what you can do to resolve it. 

  • DNA polymerase has lost its efficiency - The freeze-thawing cycles can sometimes destroy DNA polymerase’s efficiency, resulting in an incomplete PCR reaction and no PCR end-product. 
    • Solution - Use fresh DNA polymerase and repeat the procedure. 
  • Annealing temperature used was too high – Primers cannot bind to the complementary DNA when the temperature is too high. 
    • Solution – Lower the annealing temperature so it is in the optimum range. If your PCR machine has gradient functionality, perform a gradient PCR and set it to the temperature that emits the brightest desired band. 
  • Template has degraded – The cause will depend on whether you’re using cDNA or genomic DNA. cDNA is less stable and is more likely to degrade when stored at the wrong temperatures. Genomic DNA templates can also degrade because they are single-stranded. 
    • Solution – Aliquot your template DNA or run some on an agarose gel. 
  • Primers have degraded – Primers may degrade and become ineffective due to several reasons, most commonly due to inappropriate storage and frequent freeze-thawing. 
    • Solution: Aliquot your working primer solutions or use fresh stock solution to make a fresh batch and store these at -20oC.
  • PCR inhibitors are present in the template DNA – EDTA or ethanol that may be carried over from the DNA extraction process can inhibit the PCR reaction. 
    • Solution – Diluting the template DNA will reduce the amount of PCR inhibitor in the reaction allowing the PCR reaction to proceed.  
  • Contamination with DNase and RNase enzymes– These enzymes most often will break down DNA and RNA. Sometimes contamination can be from dust or dirt.
    • Solution – Clean all lab surfaces thoroughly before you start your PCR experiment. 
  • You don’t see anything during the agarose gel phase – This could be because the agarose gel dye was not added.
    • Solution – Make the agarose gel again with the dye included. 
  • PCR primers are unsuitable – One of the more common reasons for your PCR not working is because the primers are not designed correctly.
    • Solution - Redesign the PCR primers
Additional resources

Optimization and troubleshooting in PCR

Polymerase Chain Reaction (PCR)

Helixyte™ Green *20X Aqueous PCR Solution*

Gelite™ Safe DNA Gel Stain *10,000X Water Solution*