I am trying to transfect a 16kb plasmid into HEKs using lipofectamine
3000. My control plasmid is 11kb and i got some expression which was
not satisfactory with GFP (control) but none with the test plasmid. I used 2ug
(both control & test plasmid) of DNA plasmid in 25ul of Optimem and
2ul of P3000. Lipofectamine was diluted in 25ul Optimem, incubated for
5 mins room temp then mixed with diluted DNA and further incubated at
room temperature for 20mins. After which the mixture was used to
transfect cells in a 24 well plate. The genes were assayed after a 48
hour incubation.
DNA was extracted with a Qiagen Plasmid Plus Midi kit and 260/280
ratios were 1.83 & 1.86 for control and test plasmid respectively.
While 260/230 ratios were above 2.0
HEK cells were at passage number 14.
So far these are conditions that have worked the best. Following the
recommended protocol did not yield expression even with the control
plasmid.
How best can i improve the conditions?
First it seems to me that 2 ug is a bit high so the proportion lipofection and is your Qiagen Plasmid Plus Midi Kit is endotoxin free ? If not these could induce a stress within the cells that could diminish plasmid expression. Concerning the transfection, do you change your medium to serum/antibiotic free medium before transfection ?
Did you try to transfect in the same condition but with a smaller plasmid ? probably that 11 and 16 kb are too big !
If you can't improve your efficacy of transfection, try to use nucleofection system like Amaxa, they have protocol for HEK and other cells.
Abderrahim: The Qiagen kit is endotoxin free and i am using serum and antibiotic free media.
Jonathan: I haven't tried a smaller plasmid but a colleague in the department has had success with plasmids around 4kb using the same conditions. May have to try nucleofection.
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