I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all seemed to have taken the plasmid. Today, as I checked my cells, I noticed that one of them got contaminated while the other was fine. Do you know what could be a possible reason for this phenomenon? If it were the media, should not both plates be contaminated? Could there be a problem with the virus itself? My NTC plate is fine, but two of the three shRNAs got contaminated. Please let me know if you have faced similar issues with your lentiviral transfection and how you went about tackling them. Thanks! :)
This kind of problems is actually not funny and you can not trace the exact source of contamination. It happens that you touched something between your first and second plate, which explains why only your second plate got contaminated. Same theory applies when one well of your whole seeded 96-well plate!!
I would suggest to start new, unless you consider your media and virus. You can test your media on other type of cells for at least 3-4 days. You could also replace your Puromycin or check it like your media. The greatest problem is when contamination is in the virus stock. You should test it by all means and additionally for mycoplasma contamination (Very important and solves months).
Hope you solve your problem soon =D
It's really hard to track down the exact source of most contamination. And the sort of sporadic contamination you see means it's something that happened while you were handling the plates. Maybe you had something on your gloves, maybe something from the air fell on your plate. It happens.
Clean your work area really well, focus on using best-practices for minimizing contamination, practice working in a clean space (e.g. don't scratch your nose, close containers promptly, etc.). Maybe talk to a colleague that is known to have fewer contamination problems then most. They will have some good ideas to share.
Thank you Katie A Burnette and Fakry F. Mohamed for your suggestions. I agree with both of you. Apparently it was not the media because my other two plates are still growing fine without any changes to them. I have been taking extra precautions since my last contamination such as using UV light for 10 minutes before the experiment, filtering my media with 0.22 uM filter, and making sure everything is cleaned thoroughly with alcohol.
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