PCR Troubleshooting

If a standard polymerase chain reaction (PCR) does not yield the desired amplicon, it may be necessary to troubleshoot steps and optimize the reaction to attain better results. First it is necessary to assess the results. Troubleshooting PCR reactions may be a frustrating endeavor however, careful analysis and a good understanding of the reagents used in the experiment can reduce the amount of time and trials needed to obtain the desired results. In general, the stringency of the reaction should be assessed, as titration of Mg2+ and/or manipulating annealing temperatures can often solve most problems.



Below is a helpful guide in deducing issues, and troubleshooting the correct steps. At large:

Potential Issue	Troubleshooting Tips
Human error	Confirm all reagents were added appropriately at their respective steps. Ensure no reagents were contaminated or expired.
PCR reagents and additives	Prepare new reagents (e.g., fresh working stocks, new dilutions), and then systematically add one new reagent at a time to reaction mixtures to determine the culprit. Very old DNA can often accumulate inhibitors so the addition of bovine serum albumin may help alleviate the problem.
Primer dimer formation or hairpin loop structures form with the primers	Will limit the amount of product produced. Can be confirmed by gel electrophoresis, and are visible as small band on the gel of < 100 b near the bottom of the lanes. Alter the ratio of template to primer, and decrease the primer concentration if it is in severe excess to the template concentration. Add DMSO to the reaction. Use a hot start thermal cycling method instead. Design new primers.
Nonspecific products are produced	Nonspecific products can be confirmed by a ladder effect on the agarose gel and are bands that migrate at a different size than the desired product. A smear on the gel may also indicate that primers are annealing to multiple spots in the DNA outside of the target amplicon.
Concentration of Mg2+ must be adjusted	Generally, the PCR product yield will increase with the addition of greater concentrations of Mg2+, though this will also decrease the specificity and fidelity of the DNA polymerase.
Concentration of KCl must be adjusted	Longer PCR products (> 10 kb) benefit from reducing KCl from its normal 50 mM reaction concentration, often with the addition of DMSO and/or glycerol. If the amplicon is < 1000 bp and long non-specific products are forming, specificity may be improved by titrating KCl, increasing the concentration in 10 mM increments up to 100 mM.
Concentration of Deoxynucleotide 5'-triphosphates (dNTPs) is inhibiting PCR	Ensure that dNTP concentration, for each G, C, A and T is between 20-200 μM. Lower concentrations of dNTPs may increase specificity and fidelity of the reaction. For longer PCR-fragments, a higher dNTP concentration may be required. A large change in the dNTP concentration may necessitate a change in the concentration of Mg2+.
GC content is too high	Analyze the GC content in the amplicon. GC content >60% may inhibit the reaction, and may require an additive including DMSO, formamide, and dc7GTP.

Products

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References

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Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies