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PCR Pitfalls: The Devil is in the Details

Posted in: PCR, qPCR and qRT-PCR
pcr pitfalls

PCR was actually one of the first lab techniques I learned as an undergrad. Despite being sometimes labeled as a pretty basic lab skill, PCR doesn’t always work as expected. This “fickle” success is due to small details or hidden hazards within the PCR workflow that can cause your seemingly uncomplicated experiment to fail.  This wastes time and adds frustration to your day.  While I wouldn’t consider myself a master of PCR by any means, here, I describe some of the lessons I learned in identifying and overcoming PCR pitfalls.

Pitfall #1: Starting With Subpar Samples

Good beginnings make for a successful ending. Likewise, starting out with quality template DNA makes all the difference in your final PCR product. It’s nearly impossible to have a successful PCR reaction if your starting samples are not up to par. Ideally, the best DNA sample is a fresh sample. Since that’s not always possible, your best bet is a sample that has been through minimal freeze/thaw

For standard PCR reactions of 50 µl, use 25–100 ng of human genomic DNA. To prevent overloading the reaction with too much DNA leading to unspecific products, make a series of standard dilutions using your DNA and run PCR on these diluted samples to verify the limits of your reaction.

A spectrophotometer gives you an idea of your DNA concentration and quality. Looking at quality, samples with an A260/280 and A260/230 ratio greater than 1.8 are typically fine for analysis. Lower values indicate contamination. Common contaminants such as hemoglobin, ethanol, and phenol are potent inhibitors of PCR. For instance, low A260/230 values indicate contamination with salts or some solvents, while a low A260/280 value may indicate protein contamination. If you do have contamination, run a cleanup protocol, such as a commercially available clean-up kit.

Pitfall #2 Primary Sequence and Secondary Structures

When designing primers, pay attention to primary and secondary structures that gum up the reaction. In fact, it is best to avoid AT and GC rich regions altogether by choosing alternate regions farther up or downstream. Of course, this isn’t always possible such as when cloning a specific region. If you are constrained to a less than ideal region, here are some basic tips on working with these complicated gene sequences:

  • For AT rich regions, use primers longer than the standard 18–22 bp. In addition, a two-step PCR (without a separate extension step) works wonders in this case; simply remove the extension step from your PCR protocol and add that time onto the annealing step. Also try adding additional MgCl2 up to 10 mM.
  • GC rich regions, particularly when the GC content is <60% present a challenge due to the formation of stable secondary structures that stall or reduce the polymerase reaction. It might be hard to melt some GC-rich structures without extended melting periods. I’ve had great success using proprietary commercial additives to help with this. Also, some additives, such as DMSO, lower the melting temperature of DNA. Try adding 5–20% DMSO, 5–20% formamide, 1–3 M betaine or 0.01–10 mM tetramethylammonium chloride to help melt those structures.

Pitfall #3: Using the Wrong Reagents and Operator Error

If you have a standard PCR reaction that is suddenly not working, there could be an issue with the reagents. Taq polymerase, dNTPS, and your trusty primers are all subject to degradation. If you think this is the problems, try some fresh aliquots. If using new aliquots, double check that they are diluted to the correct concentration. Make sure the primer concentration is between 0.1 and 0.5 µM and dNTPs are at 200 µM.

Depending on your application, you may need to use a different Taq polymerase. If you are making a long PCR product (over 10 kb) use an appropriate long-range Taq, such as TAKARA LA. For PCR cloning, use a high fidelity Taq to minimize errors in your final sequence.

If all else fails and you know the reaction should be working but isn’t, try calibrating your pipettes. Or there is also another possibility– you may have made a human error by forgetting to add an important reagent.

Pitfall #4: Not Optimizing the Reaction Conditions

Repeat after me: there is no substitute for a gradient optimization. While the recommended annealing temperature is certainly a good starting place, you can’t just assume it is ideal. Use an annealing temperature up to 5°C lower than the primer melting temperature (Tm) to diminish nonspecific PCR products including primer dimers. In other cases, I’ve performed my annealing step at or above the Tm to allow production of my target sequence to outcompete the production of non-specific products

By using these tips, you can hopefully avoid the common PCR pitfalls that have befuddled many novice and experienced researchers alike. Best of luck with your future PCR experiments.

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Image Credit: Tim Green

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