Unfortunately, our stock of Lipofectamine 2000 got contaminated by bacteria: how can I sterilize it without losing their DNA transfection properties?
Do you have a Dos and Don't's list?
Thank you in advance,
Giulia
Hi,
You can filter with 0.2 uM filter.
If it is not worked for transfection, you can try another simple method with Calcium Phosphate.
Best,
RV
Filtration is an option. However, I would propose to discard this lot and to use a new one. Because, in further experiments and the corresponding analyses, you can never eclude a possible influence of bacterial contamination.
Rajesh Vinnakota Andreas Eisenreich Thank you! It's a very small volume (1mL approx.) and it would be almost lost in the common syringe filters: what kind of filter do you advise to use? Or what technique to recover as much volume as I can?
Unfortunately, we cannot use another vial for economical contingencies.
A small syringe filter will suit for this purpose. We can filter less than 500 uL as well.
If Possible first, try with water and practice. If you are able to filter then try with your Lipofectamine. I hope it will work.
E.g. 0.22 µm (e.g. see https://www.tpp.ch/page/produkte/12_filtration_spritzenfilter.php?lang=DE)
U can centrifuge at high speed for 5 mins.. And then do ur filtration. Do check with 1 ul in fresh media before doing ur real exp. And prewet the filter so ur lipo won't stick to the membrane. I am not sure what are these liposomes suspended in if you know you can make a sterile solution of the same and prewet the filter. Know the type of filter hydrophillic hydrophobic and low protein binding, high protein binding like nitrocellulose or PVDF
use Pen/Strep and Amphotericin/Gentamicin sol-n together, keep contamination under control, after transfection (12-24h) wash cells well with PBS, and keep antibiotics for 10 days. This will work.
Usually reagents like lipofectamine should be aliquoted and stored. You should use the vials as and when required. It's difficult to remove the bacteria completely, but you can try passing through syringe filter, once or twice. But it's better to discard the contaminated batch. Also you can use antibiotics in your Opti MEM media.
While agreeing with other comments that reagent should be replaced, I had the similar problem when my extracted DNA had bacterial contamination. So the way I did it I prepared the media/DNA/lipo solution in the tube then filtered it through 0.2um filter and added to the cells, worked quite well.
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