Co-Transfection Query - How to go about a co-transfection (Jul/12/2007 )

Hi guys,
me planning to do a co-transfection experiment and subsequently do a luciferase reporter assay. i am new to this, and could you please enlighten me on the aspects of co-transfecting 2 plasmids in hela cell lines. what controls i need and how can i check for the efficiency.
thank you
rajgene

You will have to optimise transfection with a fluorescent plasmid. Helps give a better and quick indication for transfection efficiency. then based on optimisation, transfect 2 plasmids, with the amount of DNA constant.

When you transfect always do a control transfection with a fluorescent plasmid to confirm transfection efficiency.

Think of as many controls a possible.

hi scolix,
I wanted to know what you mean by standarize transfection by fluorescent plasmid then transfect 2 plasmids.
In other words my question is will the transfection efficiency not differ when we transfect 1 plasmid and when we transfect two plasmids together.

Actually my in case my constructs are GFP and I my control is fluc. so if cotransfect them won't the trasnfection efficiency go down?
next question is -if in my cell line 3ug DNA and 6ul of LF works, then co transfection, should it be 1.5 +1.5 DNA or 3+3 DNA
thanks
mili

When you do co-transfection, always keep the total DNA dosage and lipid to DNA ratio the same across. Sometime a third unrelevent plasmid can be used to adjust the total DNA the same, while the ratio for the other two varied. For example, you can use EGFP-taged plasmid, luc construct and an empty vector combination: one well use 0.1 ug luc, 0.8 ug EGFP-tag, the other use 0.1 ug luc, 0.8 ug empty.

scolix wants you to do an EGFP plasmid-LF transfection using different DNA dosages and lipid ratios to the cell type you will use first, before even start co-transfection. This will tell you the best condition for your transfection experiments. You may find out 3+3 ug and LF may be too toxic to your cells.

thanks, I got the idea