4 replies to this topic

[Curtis]

Hi all,

I have cloned a gene into a pEGFP.N2 vector, and I am sure the gene is on the right frame as the GFP is clearly glowing under microscope ad also sequencing results are quite ok.

However the transfection efficiency of the cloned plasmid is 30% only, compared to the original pEGFP.N2 that has 60-70% efficiency.

Is it always like that? Do cloned plasmids with insert always show less efficiency?

[pDNA]

have you used an endo-free kit for the preperation of the plasmid? ...do you prepared both plasmids yourself? ...have they been isolated from the same E. coli strain?

All this factors (and more: plasmid size and so on) do effect transfection efficiencies in mammalian cells.

Regards,
p

[Curtis]

I did conventional method for cloning. I did not use any endo-free kit.
my boss says probably after gene insertion into the MCS site, the GFP gene falls way behind the promoter thus making it less expressed. He suggests culturing more cells in a bigger surface area.

[Dr Teeth]

Curtis, on Jun 24 2010, 11:12 AM, said:

I did conventional method for cloning. I did not use any endo-free kit.
my boss says probably after gene insertion into the MCS site, the GFP gene falls way behind the promoter thus making it less expressed. He suggests culturing more cells in a bigger surface area.

Are you adding equal amounts of plasmid (i.e. 1 microgram of each/well) or are you adding equimolar amounts of plasmid per well (i.e. the same number of DNA molecules).  Using equal masses is improper, yet frequently done. You should be transfecting the same number of molecules of plasmid A vs. plasmid B, which will vary unless the sizes of plasmid A and B are the same (using the same vector backbone for both can alleviate this problem). The difference between the equimolar amount and the total mass can then be completed with naked vector (assuming that you have naked vector available which does not influence your assay in any way).  Since your plasmid with insert is larger than the plasmid without, it will require more plasmid mass to yield the same number of molecules.  This obviously influences expression.

Example:
150 ng/well of plasmid A + 150 ng/well naked vector (total 300 ng/well)
vs.
200 ng/well of plasmid B (equimolar amount) + 100 ng/well naked vector (total 300 ng/well)

[Curtis]

thanks so much Dr. Teeth,
I usually transfect equal mass after measuring concentration by NanoDrop. my original vector is 4.7 kb, and the gene of interest is 1kb. I'm very happy to learn this from you. It is common mistake I guess, because I've seen many people doing it like me and nobody told me it is wrong.