transfection problem...please help... - (Jan/09/2007 )
For 7 months, I am working on co-transfection of some transcription factors and one enzyme which cuts my ligands. I am using 293T as cell type and polyethylenimine as transfection reagent. Till today it worked just 2 times. Despite everything is same, I couldnt get repeatable results. I should co transfect high amount of DNA (since there is no expression when I transfect low amount) and therefore I should add high amount of PEI. High PEI amount result in cell death.. The maximum amount that I could cotransfect was 3-4 ng per well (for 24 well plate).I am cotransfecting bgal, luciferase, 2 different steroid hormone receptors and one enzyme DNA.Without cotransfecting our enzyme DNA , the transfection with PEI works quite good.When I start to transfect enzyme DNA it doesnt work at all. Does anyone has idea? What should I do to co-transfect my enzyme DNA beside the other genes? Should I change my transfection reagent or cell type? Please help me. I couldnt cope with alone.
PEI can be toxic to cells. why dont you use calcium phosphate? it is easy and HEK are easily transfected by that.
When it is used in right condition, it is a cheap and reasonably good transfection reagent. First, try to get some LPEI from Polysciences, inc. Your next choice is from sigma-aldrich, branched form, MW 25,000. There are some more expensive ones available as well, but I would get some lipid reagents with that kind of money.
Dilute it in DW, 0.43 mg/ml. adjust pH with HCl 1N to ~7.0. Use DNA 0.2 ug /well for 96 well plate and multiply by a factor of 2 when move up to 48 well, by 4 if 24 well. Use DNA: PEI wieght ratios btw 1:0.5 to 0.8. Both diluted in serum free medium. Use OptiMEN if you have some, RPMI is good too. For 0.2 ug DNA I would use 50 ul medium and same for PEI. Add DNA into PEI then incubate 5-10 min. Add the mixture to cells with freshly added 100 ul complete medium with 10% FBS, so the final FBS is ~5%. incubate 4-8hr. try not to add directly to the cells, as 293 attach loosely to the plate, add along the side.
Try my way, you should be ok with 293 cells. The DNA list here is the total amount per well and you can use a mixture of DNA of your choice.
Gene hunter, what is the maximum amount of DNA that you co-transfect to HEK cells by PEI? Because till 1ug DNA/well for 24 well plate, there is no problem. But in order to see affect, I should co-transfect around 3 ug DNA/well (for 24 well plate)?I am using 2ul PEI for 1ug DNA. Therefore I add 3 fold PEI, which makes the high toxic affect...I am doing everything as you do. I am using serum free DMEM for dilutions and I use the PEI from sigma-aldrich.
If you are trying to transfect 3 plasmids together, try to use 0.33 ug each and limit the total DNA to about 1 ug per well and use ~0.8ug PEI (use 0.6, 0.8, 1.0 ug to find out which one is the best in terms of over transfection efficiency and toxicity.) use a reporter plasmid such as EGFP or luciferase construct to guide you in this process.
I am doing all the things that you do actually.Just in the transfection medium I use DMEM buffer without any FBS. (You said at the last volume you have 5 % FBS).
I need to transfect 5 different plasmid And one of them must be transfected in high concentration.For luciferase I use 0.45, for bgal 0.36 for two different nuclear hormone receptors totally 0.18 and for my enzyme DNA I have to use 0.3, 0.6, 1 ,1.3, 1.7 ug (in each well I have to increase the amount of enzyme DNA). Therefore totally I use too much DNA and PEI which cause cell death.In this situation do you have an idea.I am open all suggestions.Thanks in advance.
i have 22 KDa linear PEI and it is dissolved in 150mM NaCL. in the protocol they say serum doesnt effect the transfection effeciency but due to the higher survival rate, it is even better. they advice against the use of buffers. only 150mM NaCL.