Hi everyone,
I've got a problem with DNA contamination. I've extracted DNA from one cell culture and run NGS. Received data suggest sample contamination - with closely related species, also present in our lab but used in different purposes. I have no idea, where I made a mistake during isolation (CTAB-based protocol, large scale).
I used sterile, Axygen plasticware and nuclease free, DEPC treated water (autoclaved twice) to wash the cell pellets and GE Healthcare nuclease free water to resuspend the isolated DNA. The pipettes and bench were cleaned using 10% sodium hypochlorite and then 96% ethanol, treated with UV lamp afterwards (30 min of exposure). DNA extraction was performed in PCR cabinet workstation with air recirculator. Gloves were changed frequently, tips were discarded after single use. I even used a bunsen burner (in case if air circulator would be flawed).
On that day, I didn't contact with the contaminant species. Besides, the cultures are grown separately in different fitotrons under different conditions. The species are morphollogicaly unrecognizable, if one of them (contaminant) is bleached.
The extracted DNA samples of both species are stored in separate boxes, different rooms and were never put together.
Hello Natalia,
If you are absolutely certain that each step you describe was performed without contamination, one possible (and the most simple) explanation would be that your cell culture contains two or several species... I think this would be the first thing to check.
Hi, thanks very much for Your answer. Now I am uncertain of everything. Yet all that I know is our species B is photosynthetic, while species A isn't. Which is why I had to be colorblind (we don't grow bleached ones) to confuse the samples. I had no reason to consciously sabotage my own work.
I didn't check the purity of received culture via PCR with species-specific primers (I'm going to run some amplification as soon as possible). Only watched some cells under the microscope. None of them had chloroplasts.
If you can check the purity of the culture and it's OK, then its really hard to tell which step was responsible for the contamination by just reading your protocol. The person who actually performed each step is the only one who can guess what happened...
I couldn't figure out exactly what was contaminated : your final DNA extraction or PCR products ?
My prep was fine, the culture was flawed. In the presence of light a lot of cells turned green. New cultures were ordered. Thanks very much for Your suggestions.
So that was it... the simplest explanation is usually the correct one ! I hope you next culture will be OK.
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