Amplicon contamination poses a real problem for laboratories performing PCR-based experiments. Amplicon contaminants can come from any RNA or DNA, from a previous PCR amplification, or from a laboratory procedure, such as a plasmid preparation or in vitro transcription. If proper precautions are not taken to prevent amplicon contamination, the contaminants can serve as a template and interfere with your experiments by creating false positives or other undesirable results.
Here are 10 tips for preventing amplicon contamination in your lab:
Define a lab workflow. The lab workflow will look different depending on your needs and resources. If you have space, set up your PCR in a separate room from both general lab procedures and downstream processing of amplicon-generating PCR experiments. Otherwise, establish a physical workflow within the same room by denoting specific areas with signage or tape markings. Any level of one-way flow that you establish, thus eliminating backflow, will help prevent contamination.
Color code different zones of your lab. Put those multicolored tube racks to good use by organizing them around your established workflow. For example, use green for pre-PCR work, yellow for PCR setup and run, and red for post-PCR processing and analysis. Follow the same color scheme throughout, wherever possible, using tape, pipette labels, racks, etc. Use a different labcoat, fresh gloves, and safety glasses, each stored in the respective zone to help prevent carryover. Isolating anything that is touched during lab work, to a given zone, will limit contamination.
Institute cleaning procedures that match your workflow. Knowledge of the workflow should extend beyond your lab staff to facilities staff as well. Make sure cleaning personnel know the correct order to clean floors, to clean touch points such as light switches and doorknobs regularly, and to remove trash from the lab instead of rolling large trash bins through the labs, which could spread contamination.
Clean regularly with 10% bleach. It is important to wipe down any equipment or areas of suspected contamination as well as clean all work surfaces and other touch points regularly with bleach. Bleach is corrosive, so after an appropriate contact time of a few minutes, be sure to wipe those surfaces with isopropyl alcohol or water. You can monitor cleaning effectiveness by swabbing an area and testing for amplicon using PCR. Regularly make a fresh bleach solution, as it deteriorates when diluted.
Clean before you work. Use a fresh bleach solution to clean work surfaces before you begin an experiment. This is particularly important in a high-traffic lab.
Use good laboratory practices. Don’t wave a wet tip around under the hood. Use a paper towel or other clean absorbent barrier when putting a plate down that may be contaminated with amplicon. Limit personal items in the lab – for example, keep cell phones at your desk.
Use technology to eliminate contamination. You can incorporate the use of dUTP in place of dTTP in your PCR reactions and treat each PCR withto eliminate any contamination from a previous PCR1.
Don’t be penny wise and pound foolish. Use new tips for each transfer of solution or completed PCR sample as well as fresh gloves for each experiment. Store tips, plates, gloves, and other lab supplies away from your experimental setup to prevent contamination. In a shared lab, label your tips so that others won’t throw away your tips because they don't know where that half-used box has been.
When in doubt throw it out (or bleach it). This goes for tips, plates, gloves, and even paperwork - scan and shred instead of risking contamination.
Establish a written procedure. Train new lab members and provide periodic refresher training for existing members. Don’t let all of your hard work slowly slip back into “the way it’s always been done” or bad habits picked up while working in other labs.
Taking precautions to prevent amplicon contamination will minimize the need for troubleshooting and additional work. A bit of planning will go a long way!
1. Longo, M. C., Berninger, M. S., & Hartley, J. L. (1990). Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93(1), 125-128.