Great Tips for how to make the best out of your experiments
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 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.
Ideally, these two rooms should have completely independent laboratory equipment such as pipettes, centrifuges and vortexes. Ensure each area has its own protective equipment, such as gloves, lab coats and a dedicated supply of consumables. Ideally, these rooms should be ventilated by separate systems as well.
Unidirectional Workflow – A “one-way” workflow between the different areas i.e. Pre-PCR and Post-PCR is an important rule. This means that consumables and PPE (lab coats, gloves, goggles, etc.) that have been introduced into the post-PCR room would never be placed back in the pre-PCR room without thorough decontamination. Be aware that it is also possible to transmit contamination via jewelry, cellphone and even hair.
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.