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weird bacterial contamination following transfection - (Sep/26/2008 )

Please help! I'm totally stumped.

I'm doing 96-well transient transfection using Fugene HD in CHO cells (in media with serum). I've noticed 4 days later in some plates (each experimental condition was run in duplicate) there was the usual white contamination. Moreover, some plates were completely over-taken by contamination; other plates had a column that was contaminated (i.e. column 9- rows b -h). While its duplicate was not contaminated.

Any ideas on source??? the media was used to plate other cells in T-75 flasks a couple of days ago and everything looks fine there.
Also, my plasmid DNA was used previously and also yielded some contaminated plates, although not in the same wells-- it was more sporadic. It is possible that the DNA was contaminated???

The hood is continuously sprayed with 70% ETOH... and I will be cleaning the hood and incubator today with Sporicidin to remove any remnants floating around the hood. But I'd like to know the possible source of this.

Thanks.

-labrat612-

Contamination of the dna is very likely as most dna preps are done in an environment which is contaminated with bacteria. do you have antibiotics in your media?
maybe you`ll try to decontaminate your dna by heating it up with the vial closed or repurifying your dna?

also re-check your pipette tips and vials with/in which you make your trfx complex. always prepare your complex under the hood with sterile equipment.

good luck smile.gif



QUOTE (labrat612 @ Sep 26 2008, 04:45 PM)
Please help! I'm totally stumped.

I'm doing 96-well transient transfection using Fugene HD in CHO cells (in media with serum). I've noticed 4 days later in some plates (each experimental condition was run in duplicate) there was the usual white contamination. Moreover, some plates were completely over-taken by contamination; other plates had a column that was contaminated (i.e. column 9- rows b -h). While its duplicate was not contaminated.

Any ideas on source??? the media was used to plate other cells in T-75 flasks a couple of days ago and everything looks fine there.
Also, my plasmid DNA was used previously and also yielded some contaminated plates, although not in the same wells-- it was more sporadic. It is possible that the DNA was contaminated???

The hood is continuously sprayed with 70% ETOH... and I will be cleaning the hood and incubator today with Sporicidin to remove any remnants floating around the hood. But I'd like to know the possible source of this.

Thanks.

-THE_PROFESSOR-

Thank Prof for the quick reply.
I don't have antibiotics in my media-- and adding any is not an option.
Everything for the transfection (except the DNA) is prepared in the hood-- so there is a possibility of tips having something on them.

This weekend, everything was wiped down and cleaned within an inch of its life. With luck, any unwanted junk has been removed.

-labrat612-