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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:
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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.
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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. You carefully set up your PCR, excitedly waited for it to finish, ran your gel, and waited for the big reveal.