Endo-free DNA Preps, When? - For transfection?? For vaccines? (Jun/23/2006 )

For transfection?? For vaccines?

Hi,
Is enough Qiagen plasmid midi kit (or similar) for tansfections of any cell line?
I used it many times in my thesis work, but just to transfect HEK293. It worked.
Here, we use endroFree plasmid midi kit always when we need to transfect any cell line.
Endofree kit is more expensive and I think it is not necessary, but I’m not sure for all of cell lines. Does any body knows?

Second question.
We are going to assay naked DNA vaccines in some animals. In that case, do we need endro-free DNA???

For HEK293 regular prep will give you DNA, that you can transfect without killing your cells, but for other cell lines you better go for endofree DNA (U87, MT-4 are among the one I tried and endofree gave better results).

In animals, I would go for DNA that's as clean as possible, so yes, I would use endofree. Endotoxins can cause some extra reactions (immunological ones) that might alter your results, so...

Hi,

you need endotoxin free DNA for some cell lines because if you don't use it they either don't get properly transfected or they can die. For U2OS, H1299, NiH 3T3, Cos-7, HaCat, 293, Hela it wasn't a problem to use a kit without endotoxin removal solution.
An alternative to the kit you buy is from Promega: Pure yeald plasmid midiprep. It is not so expensive but has endotoxin removal solution if I remember correctly. Sigma also offer something cheaper.

... when your cells have the LPS receptors (TLR-4, CD14) you have to avoid any LPS contamination.

For example EndoTrap is a product for the removal of endotoxins. More information regarding EndoTrap you find here: www.endotrap.de