Plasmid transfection - should amount be adjusted according to insert size? - (Jul/22/2008 )

Hi everyone
I am doing a reporter assay, read out luciferase and B-galactosidase. I want to compare what happens to luciferase levels when I have various combinations of promoters and potential enhancers/repressors in my construct. My empty vector is 4.8 kb, by the time I have my longest promoter and enhancer regions in the construct is 7.7kb. Do I need to add more of the longer constructs to my transfection mixes so that the copy number getting into the cells is the same for all the constructs or will the difference be too small to worry about?

Thanks in advance for any help with working this out!!

If insert size is very different, I think you should use molar concentration other than weight.

at the same time, keep the total amount of plasmids consistant with the use of a dummy plasmid, so the DNA to transfection reagent ratio is the same.

Hi,

yes, we correct the size of plasmid. So what we do for all the plasmides:
bp of plasmid+insert/ bp plasmid = correction number.

In your case: 7700/4800= 1,6

Then, if you want to add 0,15 ug of your plasmide, it would be:
0,15ug of plasmide alone
0,15ug *1,6 of plasmide+insert = 0,24ug

We do this for all plasmides with their corresponding empty vector.
Then you have to equalize the total amount of DNA/well or plate. We replenish with pBSKTT.

lastly, also note that transformation effciency decreases with increasing size of the plasmid.