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Seven tips for avoiding DNA contamination in your PCR
Rescue your experiments from PCR contamination
Successful PCR is the backbone of numerous experiments—whether you're genotyping, creating NGS libraries, studying single cells, or testing sensitive clinical samples. If the PCR goes wrong, this can impact your entire experiment.
There are many ways a PCR experiment can go wrong, ruining your hard work. Environmental contamination of PCR samples is one such error, but it's one you can easily change.
Avoid DNA contamination with these tips
Obtaining a clean, successful PCR requires samples free of exogenous DNA. But contaminating DNA can be lurking around every corner—from previously amplified products hanging out in the lab to your own DNA. The good news is that you can largely avoid common types of contamination by following these simple guidelines:
- Designate and use distinct areas for sample preparation, PCR setup, and post-PCR analysis. To avoid contamination from old amplicons, set up the stations on separate benchtops, one for pre-PCR (for PCR reaction setup only) and the other for post-PCR (purifying PCR-amplified DNA, measuring DNA concentration, running agarose gels, and analyzing PCR products).
- Restrict equipment to these areas. Keep the PCR machine and electrophoresis apparatus in the post-PCR area.
- Prepare and store reagents for PCR separately and use them solely for their designated purpose. Aliquot reagents in small portions and store them in either location based on their use in pre-PCR or post-PCR applications. Store the aliquots separately from other DNA samples.
- Use separate sets of pipettes and pipette tips, lab coats, glove boxes, and waste baskets for the pre-PCR and post-PCR areas. If you're doing NGS library prep, use a surface decontaminant for nucleic acids to wipe down benchtops and pipettes before you begin.
- Use pipettes and pipette tips with aerosol filters dedicated for DNA sample and reaction mixture preparation.
Follow the golden rule of PCR: DO NOT bring any reagents, equipment, or pipettes used in a post-PCR area back to the pre-PCR area. This even goes for your lab notebook and pens. Label pre- and post-PCR items, so it's easy to tell where they belong.
- Keep the number of PCR cycles to a minimum, as highly sensitive assays are more prone to the effects of contamination.
Detect PCR contamination before it affects downstream applications
To check for contamination, ALWAYS perform a negative control reaction that omits template DNA and substitutes it with ultrapure water. (No band? Empty gel lane? No contamination!)
Stay tuned for more tips and tricks to get the most out of your PCR assays.
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