5 replies to this topic

[howlinjill]

when transfecting cells, say in one cell plate you put 6ug DNA and in another 10ug DNA, do you balance the difference by adding empty bluscript vector so that you are transfecting with the same amount of DNA say 10ug, all the time? How necessary is this adjustment? Why?

thanks Jill.

[fred_33]

ho
you do not have to balance this gap. these differences are generaly used to test transfection efficiency regarding the same quantity of cells in the plate.
Moreover never balance with empty bluescript. this empty vedctor will also transfect cells. even if i don't know this vector's specificities, isuppose it give an antibiotic resistance). Hence, you won't be able to select cels that get your plasmid of the cells that just get empty bluescript

[Simonsays]

I personnaly always complete the amount of DNA transfected with mock vectors. Unless you got a selection marker you are using on the mock vector but since you are transfecting cells and not bacteria, I doubt it.
The reason to do this is to get uniformity among your transfections, so everything starts equal....

Simon

[mnqcljsm]

i dont think you really need to, i dont anyways

[BryanW]

For co-transfection (2 or more plasmids) analysis and interpretation, you definitely need to use equal amount of plasmids for transfection (meaning use empty vetor to match up the difference between your transfections)......

[AussieUSA]

An alternative to mock or empty vectors to standardise the amount of DNA in a transfection, is to use sheared salmon sperm or herring sperm DNA (available from most molecular biology companies, for hybridisation).

Using a non-specific vector can often cause problems - negative interaction between the mulitple promoters and other vector elements. Therefore this is not the perfect solution for every cell / vector combination.

If you don't need to standardise the amount of DNA though, don't use anything