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.
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
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.
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.