We use L-PEI (25kDa). Prepare 10mM stock solution. First try out in 24-well plate with seeding density of 50K cells/well. Proceed to transfection after 18 hours. Use DNA 800-1000 ng/well (max). For optimal PEI amount, try out N/P ratios of 10, 15, 20 and 30. Toxicity usually starts from N/P 20 whereas N/P 15 works fine in my case. N/P calculations can be found out at following link: http://www.sciencedirect.com/science/article/pii/S1046202303003062 

Hi Norah

If your stock of PEI is 1mg/ml, use a ratio of 1ug DNA/3ug PEI (3ul) when transfecting HEK293 cells in a 6 well dish. Form the complexes at room temp for 10 minutes without serum or antibiotics. Add to cells in media (with serum but without antibiotics) and incubate 4 hours at 37 degrees/5% CO2. Remove transfection media and replace with fresh media (with serum but without antibiotics). 

Good Luck!

Hi Nora,

PEI is not a lipid mediated transfection, it is a polymer. Your best ratio will depend on what type of PEI, linear or branched, what molecular weight etc.

For your application, I can definitely recommend DreamFect Gold (lipid-based reagent). We can send you a sample if you want, just let us know at [email protected]

http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html

Good Luck

Olivier

Thank you all for your input and suggestions.  Tom yes that's the standard practice of transfection with PEI that I use but from what I've been reading that for optimal DNA clevage you need an excess of Cas9.  I transfected a 6-well plate with 0.5, 1 and 3ug of DNA at a PEI:DNA ratio of 3:1.  It's been 16 hours and half the cells and all the cells from the 1.5 and 3ug transfections are dead but the 0.5ug is fine (no antibiotics in the media).  Any suggestions as to what the cause might be ?  I know that PEI can become toxic the more amount you use so maybe that's the case since the well volume is small (2mL) ?  Normally for a 10cm I use 3ug DNA and have never had a problem  

Hi Nora,

Just so we are on the same page, you are adding the transfection mix to cells that have serum in the media (5% or 10%)? And you transfect for ~4 hours and then change the media?

Hi Tom,

yes the transfection mix I added to the cells with media + serum - antibiotics (DMEM F-12 + 10% FBS) and changed the media after 4 hours

Hi Norah,

Could there be something toxic to the cells in the DNA you are transfecting like endotoxin? Seems strange that the more DNA you add to the transfection the more dying/dead cells you see. I don't think its the PEI. I have used very high ratios of PEI when optimizing transfections and I don't really see that much toxicity. The cells look a little rough but they don't die.

The only other thing I can suggest is growing  the HEKs in DMEM high glucose media. That is what I normally use. Perhaps that will help them make it through the transfection/recovery.

I have some experience with plasmid as a jeneral