transfection gfp visualization - (Jun/26/2006 )
just wonding if people have had more luck viewing gfp plasmids in transfected cells by mounting on coverslips or just looking in dish with inverted scope??
You may get better pictures using coverslips, but typically there is no difference for EGFP signal intensity for cells on dish or coverslips.
I agree with genehunter: there's no difference in the signal intensity but it's easier and gives you better pictures to use coverslips. At least in my experience
Flow cytometry is definitely more sensitive than microscopy for GFP detection.
That's true but it takes longer and it's more expensive...
i do both - but decide depending on downstream application
i.e if i have plated all on coverslips then coverslips! but, if i am extracting RNA or protein, i don't bother with coverslips and just check the flask for transfection efficiency before proceeding
Didnt find any difference between coverslip or looking into the dish.
DNAfactory,
Flow cytometry may take slightly longer than looking down a fluoresence microscope but not that much longer. As for the expense I guess it depends on your access to flow cytometers and how much the "in house" facility charges for it. Here we're fairly lucky there are 3 or 4 dedicated to research use only and the cost is reasonable. You will pick up expression you won't see down the fluoresence microscope though.
All the best,
Ceri
I can really use a personal FACS mechine. It can provide me quantitative data.
Ceri, Can you provide me with a paid service?
Just kidding :-)
It's in the post. Might give your postman a bad back though.