There is a right way and a wrong way to set up a PCR laboratory. Because of PCR’s tremendous ability to amplify small quantities of DNA/RNA template, even the smallest of template contamination can become a huge problem in PCR. However, contamination does not have to be a problem in your laboratory. Read below to learn how to properly set up your PCR laboratory to avoid contamination. Hint: If you do not get the details right, you will have a devil of a time with your PCR reactions.
Know Thy Enemy: Amplicon Aerosols
How you should set up your PCR laboratory revolves almost entirely around avoiding PCR contamination. The single biggest source of contamination is the aerosolization of amplified PCR product. Whenever you flick open a tube of amplified PCR product, or pipette PCR product, a small amounts of your PCR product becomes aerosolized. This aerosolized PCR product or amplicon aerosol can then travel all over your bench and equipment. There it lies in wait until you assemble another PCR reaction. At which time it can become incorporated and accidently amplified – ruining your PCR and your life in the process.
Know the Proper Laboratory Setup to Combat Amplicon Aerosols
There are a number of ways to avoid amplicon aerosol contamination. The best way is to always keep your yet-to-be-amplified PCR reagents and assembled PCR reactions separate from amplified PCR products. This means that all of your PCR setups you should be done on dedicated bench space with dedicated equipment, including dedicated centrifuges, racks, tubes, lab coats, pipettes, and pipette tips, etc. Analysis of your PCR product (e.g. gel electrophoresis) should always take place on another bench, with a completely different set of equipment.
During my graduate work we did A LOT of PCR. Therefore, proper PCR laboratory setup was a priority for us. To this end we even had a separate room for PCR setups. We even went so far as to never store extra PCR tips or tubes in any place except our PCR room. Our PCR room also had a slight positive air pressure compared to the adjacent room where PCR products were handled. This was to discourage amplicon aerosols from flowing into our PCR room.
If you can afford to have a dedicated PCR room like we did – great! But many laboratories do not have this luxury. No worries, you can still do PCR. Just make sure that your PCR setup bench is far away from your PCR product bench — I am talking benches away! Consider even trading space with another lab to get some distance. Also, if possible, perform all PCR sample preparations in a laminar flow hood that is fitted with an ultraviolet light used for sterilization between setups.
Know the Proper Laboratory Practices to Combat Amplicon Aerosols
Your laboratory setup is only as good as the people that work in it. To minimize PCR contamination, you must also have good laboratory practices. This includes:
- Using clean gloves. I shouldn’t have to say this, but I see too many people not following this simple rule: You should always use clean gloves when setting up a PCR reaction! Always change your gloves if you touch anything other than your dedicated PCR bench or equipment. Did you forget a reagent and have to make a trip back to the refrigerator? Then you better grab a new pair of gloves before returning to setting up your PCR reaction.
- Aliquoting all PCR reagents. When a fresh supply of PCR reagents arrives, you should immediately aliquot them into smaller vials. This is good for a number of reasons: 1) Smaller aliquots of reagents, means less freeze/thaw cycles and a longer shelf life for your reagents. 2) If you suspect contamination in one of your reagents, you have clean aliquots to test. 3) If you do get contamination (and if you do enough PCR, you will!), you do not have to throw out your entire supply of reagent — which can be pricey. Instead you only have to throw out the offending aliquot.
- Always including negative controls. You should always include a negative control with all PCR reactions. This control is most commonly a “water blank” were water is substituted in for the DNA template. Negative controls will help you identify contamination early on, so that you can stop it from spreading.
- Using filtered water. Like many molecular biology techniques, clean water is a must in PCR. Do not used just DI water from the tap, but use sterile filtered DI water specially dedicated to PCR setup use.
- Regularly Bleaching. Why do you think the criminals on CSI are always trying to clean up their mess with bleach? Because bleach does an awesome job of destroying crime scene evidence, including DNA. So take a hint from the criminal’s playbook. Want to reduce PCR (DNA) contamination? Regularly wipe down all PCR setup surfaces and equipment with a 10% bleach solution.
- Being aware of contamination sources. Pay attention to your laboratory flow. Note what equipment comes in contact with both PCR reagents and PCR product. Centrifuges? Computers? Glove boxes? Identify all sources, and fix the overlap by dedicating equipment to PCR setup only. Some laboratories (mine was one) even go so far as to have separate refrigerators for PCR product and PCR reagent. Reducing overlap, reduces contamination.
- Using barrier tips. Barrier tips are basically normal pipette tips with some cotton-like material in them to create a barrier. This barrier prevents what you are pipetting from aerosolizing and contaminating your pipette. Although barrier pipette tips are pricier than your normal run-of-the-mill tips they can save you money in failed experiments.
- Always adding template last. The last thing you should add to your PCR master mix is your template. This minimizes the opportunity your template has to aerosolize.
- Not sharing! Do not ever borrow pipettes, tips, centrifuges or other equipment from your dedicated PCR space. As this would defeat the whole purpose of dedicated space and equipment!
- Training all newbies. Make sure that all incoming students, rotating students, and guests know the rules. A lab is only as clean as its messiest user.
Good luck and happy PCRing!