partially ligated DNA in mammalian cells

Frank O. Fackelmayer Frank at Fackelmayer.de
Thu Apr 19 10:32:54 EST 2001


Hi Dirk,

Dirk Daelemans wrote:
> 
> He,
> Does anybody know what happens if you transfect DNA immediately from the
> ligation reaction into 293 cells.  The vector is dephosphorylated and
> ligated with insert, I would like to transfect this immediately into 293
> cells. 

This won´t work, simply because the copy number of "right" ligation
product is way too low. For transfection of eukaryotes, you need
microgram amounts of plasmid (depending on the plates you are using,
e.g. 2ug for 1 well of a 6-well-plate). If you have a "normal" cloning
reaction (with some 100ng of vector and insert), and you assume that 1%
of the ligation mix will be the correct product (which is certainly too
high!), there is a factor of several thousand between the actual amount
and the amount you´d need. 

Frank




More information about the Methods mailing list