You carefully set up your PCR, excitedly waited for it to finish, ran your gel, and waited for the big reveal. 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. So, what are you to do? How do you fix this PCR contamination and avoid it in the future?
What is PCR contamination?
The biggest source of PCR contamination is aerosolized PCR products, which are created when you open a tube or pipette amplified PCR product. Once these droplets are created, being small, they travel well. These tiny droplets easily spread all over your bench and equipment, where they can find their way into your next PCR and be amplified. Now that you know what PCR contamination is made of. How do you know if you have it?
Run Negative Controls
We know we should always run negative controls. Yet, sometimes we justify not doing it. We come up with reasons like “This experiment always works!”, “I could save on reagents”, or “I do not have the time”. 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. 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.
A PCR negative control is usually just the normal PCR master mix (polymerase, primers, buffer, nucleotides) but instead of adding template, you add water. This should result in a no PCR product and an empty gel lane. Therefore, if you DO get a band, you know you have contamination… somewhere.
Identify Your Contamination Source
Where could this contamination be coming from? Well, anywhere really. 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.
1) Rule Out Your Laboratory Environment
First thing first, remove all possible environmental sources,when tracking down PCR contamination. To do this you should:
Use a 10% bleach solution or DNA-away to wipe down your:
- Bench top
- Thermocyler lid and buttons
- Unopened filter tip boxes
- Unopened sterile PCR tubes
Correctly assemble your PCR reaction:
- Wear a dedicated lab coat. You should wear a lab coat dedicated to PCR setup. This should NOT(!) be the same coat you wear when analyzing your PCR results. This coat should not go anywhere near open tubes of amplified PCR product.
- Change your gloves frequently. 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.
- Use only dedicated equipment. The pipettes, centrifuge, and vortex you use when setting up a PCR should be dedicated to PCR setup. No amplified PCR products should come anywhere near them.
2) Rule Out Your Reagents
Now that you have minimized the introduction of any new PCR contamination into your PCR assembly, it is time to check your reagents. This means systematically substituted each of your old reagents with a new (previously unopened) reagent and re-running your negative control. Whatever substitution(s) removes your contamination bands is the contaminated reagent, which should be discarded.
Avoid Future Contamination
To avoid future contamination, you should do everything above and a few more things:
- Work in dedicated space. Setup your PCR away from where you analyze PCR results. This is best done in a hood or, at minimum, benches away from where you run gels.
- Store PCR reagents and PCR products separately. In the same way that you do not want your PCR analysis to be near where you setup, you should never store PCR product and reagents together. Whenever possible use separate refrigerators.
- Aliquot. No matter how careful you are, contamination can still happen. Contamination costs not just time but also money, as you must throw out any contaminated reagents. If that reagent is new, then that can mean $300 straight into the trashcan! So, whenever possible, aliquot your reagents into smaller tubes and only work from one aliquot at a time. Not only will this help you deal with contamination, but it also prolongs the life of your reagents by reducing the number of freeze/thaw cycles.
- Store PCR tubes/tips/racks separately. Keep your PCR stuff clean by storing it well away from PCR product. Label all PCR equipment as such and tell other lab members to respect their designation.
- Don’t flick your tubes open. Minimize aerosolizing your PCR product by never “flicking” your PCR tubes open. I know it is faster to open your tubes with one hand by flicking the lid open with your thumb, but this is bad form and contributes to your bench’s contamination. Instead take your time and use two hands to carefully open all PCR product tubes.
- Use a master mixer and add your template last. The less times you handle your template, the less opportunity there is for contamination. Therefore, you should always set up your PCRs using master mixes made with (in order): water, buffer, nucleotides, primers, polymerase, and, finally, template.
- Train others. The cleanliness in your lab is only as strong as your weakest lab member. Inform others about PCR contamination, and the steps needed to avoid it. Hey, you could even print this article and share it with them.
Good luck and happy PCR-ing!
For more tips, tricks, and hacks for getting your experiments done, check out the Bitesize Bio DIY in the Lab Hub.