3 replies to this topic

[Ambinlab]

Dear All,

I tried to transfect 293s with a big plasmid (>10kb) using PolyethyleneImine. It didnot work although it worked pretty good when I transfected GFP.

Do you guys know any other way for big plasmids other than lipofectamine?

Thanks

[perneseblue]

There are a number of lipofectamine like reagents on the market.
In fact lipofectamine 2000 is actually one of the worst performing reagents. I use PolyJet and that works alot better than lipofectamine 2000.

Aside from lipofectamine, there is electroporation. It works rather well, but a require more effort in preparing the cells. There are also viral packaging, an example being the modified Sendai virus sold by the japanese company GnomONE. (I haven't used that one before, but is supposed to be able to move upto 100kb DNA )

Before I forget, are you doing mini experiments to find which is the best ratio of DNA to transfection reagent. As the plasmid gets bigger, more transfection reagent is required.

[Aimee-Jayne]

There is also another system called the Magnefect-nano from nanoTherics. High transfection efficency and high viability.

Here is a link to their webpage: http://www.nanotherics.com/

They can transfect Primary neurons with an 11kb plasmid so shouldn't be a problem for your 293's

[cpco]

May be you shoud try Calcium-phosphate-mediated Transfection, it works very well with high molecular weight DNA.