![]() ![]() Setup your PCR away from where you analyze PCR results. To avoid future contamination, you should do everything above and a few more things: Whatever substitution(s) removes your contamination bands is the contaminated reagent, which should be discarded. This means systematically substituted each of your old reagents with a new (previously unopened) reagent and re-running your negative control. Now that you have minimized the introduction of any new PCR contamination into your PCR assembly, it is time to check your reagents. No amplified PCR products should come anywhere near them. The pipettes, centrifuge, and vortex you use when setting up a PCR should be dedicated to PCR setup. If you leave your PCR setup and dedicated equipment to do anything-get more reagents, answer your phone, use a pen-change your gloves before returning to the setup. This coat should not go anywhere near open tubes of amplified PCR product. This should NOT(!) be the same coat you wear when analyzing your PCR results. You should wear a lab coat dedicated to PCR setup. Use a 10% bleach solution or DNA-away to wipe down your: 1) Rule Out Your Laboratory Environmentįirst thing first, remove all possible environmental sources,when tracking down PCR contamination. It could be coming from 1) your laboratory environment such as your pipettes, tips, hands, bench top, centrifuge, and so on, or 2) your reagents such as your polymerase, buffer, nucleotides, water, and other reagents. Where could this contamination be coming from? Well, anywhere really. Therefore, if you DO get a band, you know you have contamination… somewhere. This should result in a no PCR product and an empty gel lane. So, don’t become over confident and don’t justify your laziness, just run your negative controls because THAT is how you will know if you have contamination.Ī PCR negative control is usually just the normal PCR master mix (polymerase, primers, buffer, nucleotides) but instead of adding template, you add water. ![]() But take it from hard-earned experience: Always run your dang negative control! Without a negative control PCR contamination can lurk undetected for some time, mucking up experiments, wasting your time with troubleshooting, and slowly spreading throughout your lab. We come up with reasons like “This experiment always works!”, “I could save on reagents”, or “I do not have the time”. We know we should always run negative controls. How do you know if you have it? Run Negative Controls Now that you know what PCR contamination is made of. These tiny droplets easily spread all over your bench and equipment, where they can find their way into your next PCR and be amplified. Once these droplets are created, being small, they travel well. The biggest source of PCR contamination is aerosolized PCR products, which are created when you open a tube or pipette amplified PCR product. So, what are you to do? How do you fix this PCR contamination and avoid it in the future? What is PCR contamination? But instead of seeing what you hoped (a nice clean gel), you see a big fat mess-extra bands and, most disturbingly, bands in your negative control. For optimal PCR results, we recommend that you use separate working areas for PCR mix preparation, template addition, and performing the PCR reactions.You carefully set up your PCR, excitedly waited for it to finish, ran your gel, and waited for the big reveal.To avoid contamination from previous PCRs, incorporate an AmpErase Uracil N-Glycosylase (UNG) or Uracil-DNA Glycosylase (UDG) step prior to PCR to reduce carryover contamination.Use clean working practices to avoid template contamination.In addition, if you perform pre-PCR and post-PCR procedures in the same area, the PCR product carryover from a previous reaction can contaminate a new PCR plate and cause positive results. This can be due to a lack of GLP (good laboratory practice) resulting in random template contaminations. If this is the case, amplification of the NTC replicates should be close, as shown below, because the same amount of template was loaded onto the PCR plate.Īmplification as a result of reagent contaminationĪll reagents used in real-time PCR reactions (i.e., master mix, water, and primers/probes) can become contaminated with template. ![]()
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