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End-Point PCR Troubleshooting

End-Point PCR is a common technique in modern biology labs. Yet every run and amplification is unique, requiring some level of optimization before getting that amplification just right, which can lead to problems.

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Faint or No Amplification:

  • Short extension time: The extension time might not be sufficient for the size of your amplicon or the speed of your polymerase. When in doubt, calculate 1 min per kb

  • Wrong annealing temperature: Calculate the annealing temperature of your primers and use the lowest one for your PCR program

  • Long denaturation step: A long denaturation step might degrade your DNA. Do not use a denaturation step longer than 3 minutes

  • High (>70%) CG content on the template: Use a higher annealing temperature on your thermocycler

  • Not enough Mg2+: Use 1.5 mM Mg2+ in the final reaction tube. If you are still experiencing problems, try to optimize the Mg2+ using different concentrations

Non Specific Amplification

  • Long extension or annealing time: Try to reduce the extension or primer annealing time in your program

  • Too many cycles: Typically, the optimal range is between 20 and 30 cycles

  • Sample contamination: Try diluting your sample/prime stock to verify if there is contamination

  • Incompatible primers: The annealing temperature of your primers should be within 2 °C from each other

Smeared Bands

  • Impure dNTPs: Refresh your dNTP stock

  • Too much template: Reduce the template concentration and the number of primer extension cycles

Weak Amplification Loci in Multiplex PCR

  • Primer-primer interaction: The primers might polymerize, check their sequence and change the primers accordingly

If you plan on repeating this experiment, make sure to follow these tips to make troubleshooting easier

  • Design your primers to be between 18 and 25 base pairs, with an annealing temperature around 65 and 70 °C and within 2.5 °C from each other

  • Mix your PCR reagents on ice to avoid non-specific annealing. This also applies for Hot Start PCRs

  • Add the polymerase right before starting your run

  • Create technical repeats for your samples to exclude manipulation problems

  • Make working dilutions of all your PCR reagents and avoid repeated freeze-thaw of the master stocks

  • If you cannot find the right primer annealing temperature, try a touch down or step up PCR

Support

Hopefully by following these tips your problems will soon become a thing of the past. If you're still having issues - or have extra tips - let us know via the live chat function.