transfection efficiency - transfection efficiency (Apr/15/2005 )

hi,

many of you talked about transfection efficiency, how did you get the exact percentage of transfeced cells for plasmid transfection? by facs or any other easy method? thank you!

jxy

hi
if you want an appoximate level, you can test it under fluorescence microscopy. But if you want a really accurate method, facs will do the job.

jxy,

the method you use is dependent on the reporter you use.

If you have a construct with the gene for fluorescent proteins like GFP, then flow cytometry is fairly accurate.
If you use a luciferase gene, then you need a luminometer/luciferase assay.
Then there is B-Gal etc. which can be seen by microscopy etc.

When you don't have a reporter, then you use a detection method that analyses expression of your protein of interest on a per cell basis.

It also depends how accurate you want the results. Some people go all the way to determining the amount of DNA incorporated into the genome using a HIRT assay alongside the fluorescent/bioluminescent assays mentioned above.