Alkami Quick Guide™ for PCR

Several conditions can effect the plateau:

♦The utilization of substrates, either primers or dNTPs.

♦The stability of the reactants.

♦End product inhibition.

♦Competition for reactants by nonspecific products or primer-dimers.

♦Reannealing of product at higher concentrations which prevents the extension process.

♦Incomplete denaturation at higher product concentration.

Increasing the number of cycles does not increase specificity or efficiency of your PCR. Because the plateau effect encourages nonspecific amplification.

Improve specificity: Reduce number of cycles. Reduce cycle segment lengths.

Improve efficiency: For amplifying large fragments (> 1 kb) increase the duration of each thermal step.

Reaction Volumes

Standard Range: 20 - 100 µL in 0.5 mL microcentrifuge tubes.

5 µL reactions have also been successful. Large volume samples will be inefficiently heated and cooled, while smaller reaction volumes will change temperature more quickly but generate less product yield. The geometry of the reaction mix tube, thickness of the tube wall, and the particular thermocycler (some models have a slow response to the heat block so that wells near the periphery are slow to heat up) are factors which influence the time needed for each of the cycle steps.

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In addition, some thermocyclers are calibrated by block temperature, not sample temperature. Others are calibrated by the sample temperature in which case you can use thin walled tubes & cut the sizes down.

Contributed by Denise Rubens, National Cancer Institute, Frederick, MD

Improve efficiency: Increase your reaction volume (as well as your incubation times to insure adequate thermal equilibrium). Use an oil cap to reduce evaporation and internal condensation (for a 100 µL reaction volume).