DNA size related to the transfection efficiency？ - (Apr/03/2007 )

hallo,
do u think the size of plasmid DNA have influency on the transient transfection efficiency ? Does the larger DNA more difficult into the cell during transfection ?
appreciate your responze

I have done transfection with plasmid as large as 15 kb with no problem. You do need to consider less copy number delivered for large plasmids when same amount of plasmid in ug used.

Do transfections with > 10 kb routinely, works nice (in 293T and Hela I've done it), but indeed consider that you got less copies than smaller plasmids with same amount of DNA.

the large the plasmid is the less copy number that you will have for the same amount of plasmid in ug, right?

do you mean every plasmid should take less copy number of gene ?
sorry , my brain cant run so fast

1 µg of plasmid of 5 kb will contain 2 times the number of individual plasmid molecules compared to the same amount (1 µg) of 10 kb plasmid, just because the 10 kb plasmid weighs twice as much.

Compare the number of plasmid molecules in 1 ug DNA for 3 kb plasmid and 15 kb plasmid, which one has more plasmid molecules? You may have low transfection rate simply due to you incorporated less number of plasmid molecules to cells if you are using large plamsids than uusing smaller plasmids. That is what we referred to.

Both of us have no problem transfecting cells with large plasmids as big as 10-15 kb in size.

How large is your plasmid in kb?

hello , genehunger (just kidding)
please forgive my responze so late, and i can catch you fully,thanxxxxxxxxxxxxxx
my plasmid is about 12Kb , so according to your experience , the size is not a question
today , i notice a problem, the cells of 293 in the well is quite different from the cells in the plate , but both are not contaminated , so do u think what is the reason ,in my oppinion the cells may be overtreated with trypsin when i plate cells into the well

I had my meal loaded with the genes that I just huntered. hmmm...


Seriously,

Does the medium look clear, not turbid to you ? if so contamination is possible.

Did you see a lot of dead, floating cells? overtreatment lead to cell death. Trypson-EDTA up to 3 min is enough for these cells.

Did you seed them at the same density (cells/cm2) for plates and dishs/flasks? Sometimes, they look differently when grow at different densities.