qPCR, with its high sensitivity, allows researchers to produce millions of copies of a specific DNA sequence from only a few initial copies. While this sensitivity is very useful, it comes with a necessity to avoid contamination of the reaction.
Contamination and non-specific amplification can cause misleading results, such as false positives and masking changes between the control sample and the tested sample. When working with plasmids, the problem of contaminations becomes even more severe and difficult to deal with.
DNA Contamination cannot be reduced or removed once occurred. Therefore, it is essential to adopt adequate laboratory practices when performing qPCR experiments.
How do we know if we have a contamination is our qPCR experiment?
One of the most common ways to monitor for contamination is to use “no template controls” (NTCs). NTC wells contain all the qPCR reaction components such as primers, master mix and water, except for the DNA template. If amplification is observed in the NTC wells, it is possible there is a contamination in one of the reaction components.
How to avoid contamination
- Pre-PCR: Optimally, this room should be further divided into two areas-
- Post-PCR: this room (or bench) should be physically separated from the pre-PCR room, designated for post amplification steps and analysis. In this area you will want to locate the qPCR instrument and perform all steps which require open tubes manipulation after PCR amplification.
1. Aslanzadeh J (2004) Preventing PCR amplification carryover contamination in a clinical laboratory. Ann Clin Lab Sci 34(4):389–96.
2. Nolan T, Huggett J, Sanchez E (2013) Good practice guide for the application of quantitative PCR (qPCR), LGC.