PCR Troubleshooting Tips

As with any experiment, problems are bound to occur. To address these problems head-on, it’s critical to develop a process for detecting potential sources of error. The polymerase chain reaction (PCR) is a widely used technique for amplifying specific fragments of DNA. With its apparent simplistic protocol and low cost, PCR is a widely adopted method. That being said, problems can—and will—occur, and knowing the right steps to solve the problems is necessary. When developing a PCR troubleshooting protocol, it is important to be aware of any possible sources of error, however insignificant they may seem, and explore each potential problem independently. Most importantly, troubleshooting is like any skill; it improves with practice and experience. Below are some tips for developing a protocol for PCR troubleshooting.

Choosing the Right Master Mix

Complications with the master mix blend (containing all reaction components except DNA target) may cause amplification failure in all samples and positive controls. Here are a few tips for finding the right master mix for your experiment.

• Before repeating the experiment, all components should be checked along with the concentrations.
• If a new batch of reagent is being used, it is a useful precaution to run the new against the old before launching into a major series of experiments.
• When switching master mix products, it is critical to recognize that some assays are particularly sensitive to buffer composition/annealing temperature (T_a)/primer concentration combinations. Changing any one of these may result in different performance. Therefore, all assays should be verified in selected master mixes and on all desired instruments before making radical changes.
• It is also essential to review the instructions provided with each master mix since these specify the recommended conditions that are optimized for the given enzyme, hot start mechanism and buffer components.

It is good laboratory practice to ensure that sufficient reaction master mix is prepared for all samples that are run together. Ensure that all components are carefully thawed and mixed well and that the experiment master mix is very well mixed before aliquoting to samples. This is particularly relevant to 2× buffers that are more viscous than normal PCR buffers.

Pairing with the Right Oligos

Oligonucleotides intended to hybridize with the target DNA and be amplified by PCR are referred to as primers. In order to anneal, primers have a reverse compliment sequence of the target DNA. Upon receipt of a lyophilized (freeze-dried) primer it is critical to first:

• Verify the sequence
• Ensure that all DNA is re-suspended prior to use
• Confirm the solution is the concentration expected

Repeated cycles of freeze thaw can also affect oligo performance and therefore all oligos at a stock concentration (usually 100 μM) should be aliquotted and stored at –20 °C or –80 °C.

During the troubleshooting phase, it is critical to verify that the correct sequence was ordered by returning to the target sequence and confirming that the oligo sequences are indeed present. Ensure that the oligo quality was correct by contacting the oligo vendor. Measure the working concentration of the oligo and visually inspect fluorescent molecules to confirm that they are labeled. Test the primers of probe assays in a SYBR^® Green I qPCR mix to verify amplification. Consider optimizing primer concentrations or T_a. When using a probe for the first time, collect fluorescent data for as many potential wavelengths as possible so that any potential leakage of signal between channels is observed and mistakes in labeling can be detected.

Optimizing Assay Design

When troubleshooting an assay, ensure that the design has been verified. Confirm that the PCR/qPCR primer and amplicon position is consistent with the reverse transcription priming protocol. Ensure that the sequence information is reliable and that appropriate splice variants and SNPs have been considered. If the assay is insensitive and the amplification plots look irregular make sure to:

• Check assay design
• Check background fluorescence readings on raw data/multicomponent plot
• Verify optimization

Remember assay optimization and validation is an essential first step, even when assays have been predesigned and commercially obtained.

Concluding Results

In addition to basic PCR protocols, a troubleshooting protocol should be developed and used when problems arise. Furthermore, to protect against uncertainties due to reaction variability, it is important to include a series of controls alongside all samples in any experiment. The choice of controls and whether to include them should always depend only upon the nature of the experiment.