When you’ve conducted the same reaction a dozen times and the problem is still elusive, it’s easy to get frustrated. Simple mistakes can turn a polymerase chain reaction (PCR) reaction into a “pipette, cry, repeat” (“PCR”) situation, but simple mistakes have simple solutions. Previous articles on troubleshooting problematic PCR have covered nonspecific binding, the absence of bands, weak bands and smearing. If none of these valuable tips have helped, the problem might not be part of your experimental process – your materials, equipment or technique may be to blame. We'll divide the possibilities into manageable categories to make it easier for you to isolate the problem.
- Maintain your DNA sample and primers to avoid a weak template.
- Quality: All substances should be properly thawed and mixed prior to use. Because repeated thawing can also damage substances like dNTP and older DNA stocks, a new solution might remedy a weak amplification result.
- Quantity: Template concentration, if too high, can create non-specific band amplification or smearing. Primer dilution can also affect success rates. After encountering such results, try parallel reactions with different concentrations of these substances.
- Contamination: Preventing contamination in your sample is easy if you’re following safety protocol. Minimize contact by wearing gloves, tying long hair and not putting your face too close to the chemicals you’re working with (these are also good rules to follow in all lab activities).
- You need an enzyme for the PCR to produce amplified DNA.
- Weak reactions: Increase or change enzymes if the one you’re using seems defective or ineffective on your sample.
- Inhibitors: Your enzyme won’t work as well if it encounters substances that challenge it, as in templates which aren’t highly purified. Adding more enzyme should obtain a better yield in this circumstance.
- Taq polymerase: Don’t accidentally use TaqI, a restriction enzyme. This isn’t going to help you whatsoever.
- Check over the buffer and any additives.
- Label accuracy: Review labels for each chemical. Ensure they are legible and the substance’s title and formula are what you expect. If someone has written what you believe is “dNTP” on a bottle in cursive, ask to be sure it isn’t actually “dUTP.”
- Buffer and MgCl2: Buffers differ, but they all work to help polymerase activity. If MgCl2 is not already in the buffer, add it to enhance extension. If MgCl2 is included in your buffer, don’t add extra, as this reduces specificity.
- Additives: DMSO and BSA are popular to foster cleaner results. While they give the PCR a helpful nudge, use additives consciously and at the correct quantities. Being cautious of incorporating too many substances makes it easier to identify where your problem is coming from.
- When all else fails: If no other substances are setting off the reaction, check the water. Make sure it has the proper pH to foster a reaction (you never know).
- Apply the proper pipette.
- Calibration: Set your pipette at the necessary calibration, using the right pipette depending on the quantities you need. Using the wrong pipette for your amount (going too high or low in the gauges) will lead to inaccurate amounts and damage the pipette’s mechanism.
- Correct tips: 20 μl and 200μl pipette tips are the same, but 1000μl models require a larger size. Don’t try to use the wrong tip for your pipette size. Investing in filter tips will also help prevent general contamination in your PCR.
- Fix it if it’s broken: Old or poorly used pipettes can fail you when you don’t notice. Check that your pipette you’re takes up and dispenses the correct amount of fluid with each use.
- Give your thermocycler a once-over.
- Settings: Thermocycler temperatures and time settings should be correct to anneal the specific sample. If temperatures are too low, non-specific priming will prevent bands from presenting; if they are too high, priming will not occur at the desired sequence. Check for effective temperatures by testing a range incrementally.
- Evaporation: To prevent sample evaporation, your thermocycler’s lid temperature should be set above the sample’s to prevent material from being lost. The seal should be firm on the tube lids to prevent boiling substance from escaping. Thermocyclers have inserts adjustable for tube shape (domed versus flat caps) to hold them shut.
- If you can’t take the pressure/heat...: The thermocycler should heat and apply pressure evenly for consistent readings to all wells; you’ll know if this is a problem when only certain locations of wells are wrong every time.
- Review data-processors.
- Machines: Instruments for real-time PCR analysis have lots of parts – detection systems have complex light and detector mechanisms which can break or wear down over time. Replacing these could make your absolutely dysfunctional PCR functional again.
- Programs: Whatever software you are using to analyze data, check that its operations are fully functional and apply to the results you’re working with. If setup, data collection or analysis aren’t programmed for your specifications, the instrument can’t do its job.
- Organization and proper use of necessary materials will optimize your PCR.
- Organize: Have the things you use lined up and ready for operation before you begin so they aren’t forgotten or lost in your time of need. Employing boxes for bottles, tips, strip tubes, etc. will make you feel (and look) ready to succeed.
- Use your materials: Having a used-unused system will keep you from second-guessing which steps you’ve already performed. Moving things to the side only after they’ve been incorporated creates a reliable system to eliminate this type of doubt. One helpful product for the occasional lapse is Goof-Proof™ qPCR Master Mix, which uses a dual color system to keep track of when template has been applied.
- Follow pipette protocol.
- Tip tips: Apply the tip with enough but not too much pressure. Insert it fully into fluid to prevent any difference in quantity being taken up, avoiding replacement of liquid with air. When retrieving the substance, don’t push past the first stop.
- Dispensing: Ensure that uniform amounts of fluid are dispensed by doing it slowly and precisely, going past the pipette’s first stop to expel residual liquid. Don’t pipette sideways and withdraw the tip from your well before releasing the button again.
- Keep it clean: Dispose of pipette tips with every use in a new liquid or after touching another surface. This prevents contamination that jeopardizes your PCR.
If you remember these material, equipment and technique tips, you’re less likely to encounter confounding problems with the PCR reaction you perform. Refresh your knowledge of protocol and guidelines for troubleshooting by revisiting the previous articles:
Laboratory for Environmental Pathogens Research Department of Environmental Sciences University of Toledo. (2004, December). Polymerase Chain Reaction (PCR). Retrieved July 19, 2017, from http://www.eeescience.utoledo.edu/Faculty/Sigler/V...
Shears, J. (Director), & Dalgleish, R., Suter-Giorgini, N., & Kramer, C. (Producers). (2009, June 25). Using a Micropipette - University of Leicester [Video file]. Retrieved July 19, 2017, from https://www.youtube.com/watch?v=uEy_NGDfo_8
University of Washington. (n.d.). Polymerase Chain Reaction Protocol. Retrieved July 19, 2017, from http://pga.gs.washington.edu/protocols/pcr_protoco...
GoldBio Staff Writer
Megan Hardie is an undergraduate student at The Ohio State University’s Honors Arts and Sciences program. Her eclectic interests have led to a rather unwieldly degree title: BS in Anthropological Sciences and BA English Creative Writing, Forensics Minor. She aspires to a PhD in Forensic Anthropology and MA in English. In her career, she endeavors to apply the qualities of literature to the scientific mode and vice versa, integrating analysis with artistic expression.
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