Does anyone have experience in transfecting a Cas9 plasmid containing the target guide insert into HEK293 cells via lipid mediated transfection (PEI)? I'm using the PX458 vector and my insert is 20 nts. I've found a few different articles that recommends the DNA:PEI ratio of 1:4 and culturing with no antibiotics. Any advice or input would be appreciated. Thanks !
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 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.
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