PCR Tips

General tips

  • When collecting tail tips, clean tools with 70% ETOH between animals.
  • Use filtered pipette tips.
  • Avoid repeated freezing and thawing of nucleotides. Aliquot small volumes and store at -70°C.
  • Do NOT perform PCR in a ventilated hood as it increases the risk of cross-contamination.
  • Mix the reaction tube by gentle tapping. Do not vortex PCR mix.
  • Add DNA polymerase (Taq) to the reaction tube last.
  • Adjust electrophoresis voltage and run time to improve band resolution.
  • Confirm that the PCR machine was programmed correctly.
  • Avoid overloading PCR products into the gel; this may result in cross-contamination or misinterpretation of the results.

Genotyping protocols for JAX® Mice are provided as a starting point for developing your own assay.

An assay that works well in one lab may not work well in another. Conditions and components need to be optimized for your lab conditions. In some cases, you may need to optimize extensively or develop a new assay to obtain acceptable results under your lab conditions.

Alter the amount of DNA per reaction.

Too much or too little DNA will result in poor amplification. Run a dilution series of DNA samples including a non-diluted sample, and samples diluted at 1:10, 1:100, and 1:1000 with H2O. If you know the concentration of each DNA solution, a 25μl PCR reaction typically requires ~5ng of highly-purified DNA or 40-50 ng of quick prep ("dirty") DNA (see DNA isolation protocols for preparation methods).

Test 'Touchdown cycling' as an alternative for multiplex PCR reactions.

Touchdown cycling is a common feature on many thermocyclers (consult your manual for instructions). This feature alters the temperature incrementally during the annealing step to accommodate primer pairs with different optimal annealing temperatures.

If multiplexing a reaction is ineffective, separate primer pairs.

If one PCR product is weak or absent in a multiplex reaction, run separate PCR reactions for each primer pair and optimize them independently.

Dilute fresh PCR reagents and/or perform a new DNA isolation/dilution.

If there are unexpected changes in your PCR results (especially in controls) or if contamination is suspected, obtain freshly-made reagents.

Use a different DNA isolation protocol.

For most PCR protocols, a quick, "dirty" DNA preparation is sufficient, but some assays improve when highly purified DNA is used. See DNA isolation protocols.

Primers

Double-check primer sequences if control bands are absent.

If the sequence is correct, consider ordering new primers if degradation or hidden production errors could be the problem.

Consider designing new primers or a new PCR assay.

Genotyping protocols for JAX® Mice are provided as a starting point to develop your own assay. References by the primary investigators who donated the strain typically contain the most extensive genetic information needed to develop new primers and protocols. Primary references are found on all JAX® Mice Database strain data sheets.

Non-specific bands

Increase or decrease the annealing temperature.

If the expected bands are not amplified, dropping the annealing temperature can help primers bind more efficiently. If bands are weak, try using a longer extension time.

If there are more bands than expected (non-specific bands are amplified), raising the annealing temperature can help eliminate non-specific primer binding.

Try a 'Hot start' to reduce non-specific bands.

Pre-heat the thermocycler to 94°C before adding samples, or use a hot-start-specific Taq polymerase (available from most companies that sell Taq), or use a Taq polymerase antibody in the reaction mixture according to manufactures instructions (i.e. TaqStart, BD Biosciences/Clontech, stock# 639250). A 1:1 TaqStart antibody to Taq DNA Polymerse ratio is commonly used for a hot-start procedure.

Use Perfect Match® PCR Enhancer to increase specificity.

This reagent destabilizes mismatched primer-template complexes to reduce PCR artifacts, thereby promoting amplification of the correct PCR products.

To reduce false positives in neo genotyping assays, use new reagents and clean equipment (filtered pipette tips, clean pipettes, a clean lab bench space etc.).

Neo contamination is common and can lead to false positives when genotyping mice using neo-specific primers. You can also develop a mutation-specific PCR assay that uses mutation-specific (rather than neo-specific) primers.