7 replies to this topic

[Baars01]

Hi,

There seems to be slight bacterial contamination present in one of our transfection agents (Lipofectamine RNAiMAX). This stuff is quite expensive, so it would be a shame if it would go to waste. I was thinking that perhaps centrifuging it at >10000g for 10 min or so and transferring the supernatant might remove most contamination. Alternatively, using a filter might do the same.

Does anybody with more expertise know what a good option is?

Many thanks

[leelee]

SOOOOO not worth it. Just buy some more.
Imagine if you got rid of the majority of the contamination so that it was only present at below detectable limit- transfect your cells, then over time contamination returns......... the time you will waste far outweighs the cost of a new kit.
If you must- then I would say filtration is the way to go (if that is ok for the components of the solutions) as centrifuging to get rid of "most" of the contamination is just dodgy!!!
It sucks when this type of thing happens!!

[genehunter]

I dont know how is that possible, as most cationic reagents have broad, non-specific antibacterial activities.  

A good spin should help.  Filter is probably not the my choice considering possible losses either sticking the membrane or due to dead space.

[Baars01]

genehunter, on Aug 17 2009, 06:31 AM, said:

I dont know how is that possible, as most cationic reagents have broad, non-specific antibacterial activities.  

A good spin should help.  Filter is probably not the my choice considering possible losses either sticking the membrane or due to dead space.

That's what I thought too, how can bacteria survive in this stuff?? My only logic dictates that it might be dormant bacteriospores that might have contaminated the transfection agent. Would centrifuging not separate the emulsions in the lipofectamine or does it only become an emulsion in aqueous solution (media)?

Thnx

[genehunter]

Double check it by adding transfection reagent  to the culture medium and use the medium alone as control.  I doubt it very much because at least it does not support the growth of bacteria, in addition that you keep it at 4 C, so you will not get much bacteria to begin with.


For your question, these are probably micelle/liposomes and you cant readily pellet these particles down through regular spin condition.

[Baars01]

genehunter, on Aug 17 2009, 07:14 AM, said:

Double check it by adding transfection reagent  to the culture medium and use the medium alone as control.  I doubt it very much because at least it does not support the growth of bacteria, in addition that you keep it at 4 C, so you will not get much bacteria to begin with.


For your question, these are probably micelle/liposomes and you cant readily pellet these particles down through regular spin condition.

Sorry for not mentioning this earlier, but I have already performed this. In duplicate, I have tested the siRNA, media, tubes etc.. all components of the experiment. Only the transfection agent comes up positive for contamination.

[memo]

Try a new agent is my suggestion as you're not sure if your values at the end of your experiment are reliable. Why not try a cheaper agent? I did some small competition experiments and found saint-red the most reliable agent with the best results.

[Baars]

Well, the centrifugation didn't work. Will probably end up ordering more of the stuff. Thanks for the advice