transient transfection

Courtland Yockey yockey at dcsmserver.med.sc.edu
Wed Feb 26 20:51:11 EST 1997


PETERSON LAWRENCE wrote:
> 
> I need help trying to figure out why my transfections aren't working
> very well.  I am using NIH 3T3 cells and Lipofectin.  I have tried
> various ratios of Lipofectin:DNA, various times of incubation, with
> Optimem or with other serum free medium.  I am currently just
> transfecting a reporter vector (pSVB-Gal) and then using an X-Gal assay
> to identify transfected cells.  Way less than 1% of the cells are
> getting transfected.  Everyone says these cells are so easy to transfect
> and I should be able to do it blind folded.  What am I doing wrong?
> 
> I start with cells that are 60 to 80% confluent and then do my assay
> either 24 or 48 hours later.
> 
> Any insights would be greatly appreciated.
> 
> Rebecca Slayton
> email: rebecca-slayton at uiowa.edu


In the case of Clonfectin (a cationic lipid reagent), the manual 
suggests using polystyrene tubes to prepare the reagent and DNA.  Are 
you using polypropylene microfuge tubes or polypropylene snap-cap 
tubes to prepare the reagents?  If so, you might consider trying 
polystyrene -- I'm not sure if this is the root of your problems, but 
it might be worth a try.

Courtland Yockey
yockey at dcsmserver.med.sc.edu



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