Advise for transfection efficiency - (Oct/24/2008 )
I transfected my CHO with two plasmids, with Turbofect from Fermentas, it is a solution of cationic polymer that forms positively charged complexes with DNA.
I have got some CHO cells stable transfected with the receptor I trasfected (2 subunits, so two plasmids) (by another lab) that I use as a control.
Well, two days after transfection I measured the expression of the receptor with citofluorimetre (cells were incubated with moAb for the receptor), and this is the result: 94% of control CHO cells (the ones stable transfected with the receptor) express the receptor; 0,06% of my Mock cells express the receptor (so, 0%); 20% of my transfected cells with wt receptor express the receptor; and (the absurd date) 5% of my transfected cells with mutated receptor express the receptor..... I supposed 0 or 20... but not 5!!
Now, I think it's a transfection problem....
First of all, how can I increase transfection efficiency? 20% isn't so good...... I used 0,5ug plasmidA and 0,5 mg of plasmid B in a 24 well plate (about 250000 cells) with 2ul of Turbofect, according to turbofect manual. I think I will increase plasmid amount, I don't know if I must increase also Turbofect amount.... what do you think?
And I will do my experiment in duplicate, so I can see if it's a transfection problem....
I have got some CHO cells stable transfected with the receptor I trasfected (2 subunits, so two plasmids) (by another lab) that I use as a control.
Well, two days after transfection I measured the expression of the receptor with citofluorimetre (cells were incubated with moAb for the receptor), and this is the result: 94% of control CHO cells (the ones stable transfected with the receptor) express the receptor; 0,06% of my Mock cells express the receptor (so, 0%); 20% of my transfected cells with wt receptor express the receptor; and (the absurd date) 5% of my transfected cells with mutated receptor express the receptor..... I supposed 0 or 20... but not 5!!
Now, I think it's a transfection problem....
First of all, how can I increase transfection efficiency? 20% isn't so good...... I used 0,5ug plasmidA and 0,5 mg of plasmid B in a 24 well plate (about 250000 cells) with 2ul of Turbofect, according to turbofect manual. I think I will increase plasmid amount, I don't know if I must increase also Turbofect amount.... what do you think?
And I will do my experiment in duplicate, so I can see if it's a transfection problem....
some cells are difficult to transfect; CHO are often used, so screen manufacturers data for alternative transfection reagents;
if your difficulties are not to circumvent by other transfection reagents, a combination of electroporation and transfection reagent may help