How to improve transfection efficiency? - (Sep/17/2007 )

Hi, all,

I am transfecting a gene (~ 800bp) to HepG2 cell. It’s never been successful. I have tried to transfect the same plasmid to another cell line N2a. Although not in high efficiency, about 30% of N2a cells can be transfected. Then I test HepG2 with another 2 plasmids, they are fine. Does anyone have advice how to improve this situation? Thanks a lot!

some plasmids are tough to transfect. Also i'm more guessing about contaminants like ndotoxin.
I was wondering if you adjust DNA concentration regarding its size against a plasmid.
I mean a plasmid is roughly considered as a 4.5kpb. So if your gene is 800bp, and as supposing you transfect either 1µg of plsmid or 1µg of the genee, isn't thee a possibility that such a high amount of a single gene becomes toxic for the cell and trigger an unspecific silencing response?
Did you monitor step with GFP gene alone to see if transfecting short sequence has different requirements? i think that worth it.

Hi, Fred,
Thank you for the answer. If 1ug of DNA needed, I will transfect 1ug of plasmid, not 1ug of inserted gene. Is it correct? And it is a good point to monitor transfection using the same plasmid with GFP only. In my previous experiment, I used a different GFP plasmid as transfection control.
Thank you, I will try it.

ps, forget to mention, when I transfect the same plasmid (pIRES-hrGFP) containing another gene (size is similar, around 800bp) to HepG2, it works well. Does it mean HepG2 doesn't like that gene?