Dear Researchers
I hope you and your research is well. My own research is a little poorly. My Professor suggested I ask independent researchers about my lab-related problems. If you could answer the following I would be truly grateful.
I am developing a method to use environmental DNA (eDNA) to detect an aquatic macrophyte in lochs.
In the PCR lab, tissue samples were extracted, with a step that included the grinding of dried material in an open Eppendorf tube using a micro pestle.
During primer design, primers were tested using standard PCR. After amplification, the plates were unsealed, and processed using ZAG capillary electrophoresis to determine nature of amplicons of target and non-target species.
Once primers had been shown to be species specific, a probe was purchased and field samples were processed using qPCR (MGB-Probe with FAM Dye, and Environmental Mastermix). All samples, and many Non Template Controls (NTCs), showed contamination.
The samples had been extracted in a room adjoining the same lab where PCR amplification occurred, after primer design. Also, all pre-PCR work (preparing qPCR plates) occurs in this side room. A sliding glass door with a 20 mm gap separates the two rooms. 20% bleach was used for decontaminating surfaces.
I hypothesised that DNA contamination was from the air. In 2 repeated experiments that exposed NTCs (no template) to the air for different time periods. In both repeated experiments, I found that no contamination (0/12) occurred when wells were sealed immediately, but 5/12 replicates showed contamination when left exposed to the air for 30 minutes. See attached picture.
Questions
1. From my NTC experiment's results, can it safely be concluded that the air is the source of contamination?
2. What lab process or experimenter behaviour produced the contamination?
3. Could contamination be minimised by using qPCR and SYBR-Green Dyes to develop primers (e.g. you don't have to uncover the post-qPCR wells)?
4. If a hood with UV lighting had been installed in the adjoining room where sample extraction and plate preparation occurred, could this have prevented/minimised the contamination?
5. What steps, protocols and equipment is used in your eDNA lab to minimise contamination?
Many thanks
Nick Crutchley
Thank you Waseem and Carin
Both answers are invaluable.
I remain doubtful that the barrier tips and reagents are to blame. The exact same tips and reagents were used for both treatments. The only significant difference was exposure to the air. 0 Contamination events when wells were sealed immediately with strip caps, 5 when left exposed. However, I remain open to alternatives to air-borne contamination, despite feeling the opening of post-pcr plates for purposes of capillary electrophoresis (we do not use SYBR Green to analyse melt curves) is causing the aerosolisation of DNA particles to spread throughout the lab.
I value your input regarding uv lightning, and the fume hood and laminar flow hood (the latter of which I believe has just been fitted, together with UV lightning). Indeed, when running an experiment in the fume hood, the rate of contamination was an order of magnitude less.
And yes, I have argued for the purchase of a closed-system tissue lyser from the beginning.
This contamination is quite tricky. Now looking to find statistical methods of piercing the background noise.
Thanks again
Nick