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Cloning PCR fragments

DAVIS at licre.ludwig.edu.au DAVIS at licre.ludwig.edu.au
Mon Aug 30 08:24:14 EST 1993


In article <25j2t7$2qb at scuba.sura.net>, inet3 at scuba.sura.net (Internet Guest3) 
writes:
> Has anyone out there used BRL's Lipofectamine Reagent for
> transfecting eukaryotic cells?  

I have used their Lipofectin reagent, as opposed to their Lipofectamine.  
It works nicely, but what the package insert neglects to mention is that 
you need a ratio of about 5:1 lipofectin:DNA to get it to work.  I use it 
at 10 mcg/ml (= 10 microlitre/ml) lipofectin to 2 mcg/ml DNA as the end 
concentration.  This needs to be optimised for the cell line and the 
plasmid  you are using, and also the time of transfection needs to be 
optimised; an hour either side of the optimum can make the difference 
between success and failure.

Somebody else at our Institute (who introduced me to Lipofectin) has done a 
few preliminary experiments with Lipofectamine and did not generate any 
colonies, but maybe it just needs a bit more tweaking.


Ian Davis                                       davis at licre.ludwig.edu.au
Ludwig Institute for Cancer Research
Melbourne Tumour Biology Branch
Melbourne, Australia.



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