Toxicity of Transfection -> Endotoxin? - (Nov/13/2008 )

I've been doing transfection of primary (fetal and adult) hepatocytes isolated from mice

I use Lipofectamine 200 and my experience is they are (surprisingly) not toxic to my cells

When I transfect with siRNA (purchased from ambion), they do fine....

But then when I transfect with vector, I saw a lot of cells died (I think 50-80% mortality, compared to siRNA transfected)

Someone told me it's the endotoxin in my vector that's killing the cells...

This is because I expand the vector in E coli and the endotoxin from bacteria is still in there after midi-prep


My question is:

I used Qiagen midi-prep kit (the one that doesn't come with a concentrator and requires centrifugation to precipitate the DNA myself after isopropanol), is that the reason I have endotoxin?

I don't have the kit that comes with a concentrator, but do I need one? Or there are other methods (without using a new midi-prep kit) to handle the problem? For example use a certain column to get rid of the endotoxin....


Thanks!!!

Use an Endofree plasmid prep kit from Qiagen.

http://www1.qiagen.com/products/Plasmid/QI...aspx?ShowInfo=1

It is possible to purchase just the endofree buffers and use them with your normal midi kit. They reduce the amount of endotoxins almost 1000-fold compared to the standard protocol.

Best,

Ginger

Ooh... just found a few kits that might be useful for you...

http://www.biocompare.com/Articles/Technol...13/Article.html

and

http://www.biocompare.com/ProductListings/...t-listings.html

Enjoy!

Ginger