hoffmann at chem.vu.nl
Mon Apr 29 04:26:54 EST 1996
Ian A. York wrote:
> (I've asked this before with no responses, but I'm getting frustrated and
> will try again)
> I'm trying to get efficient plasmid extraction out of transfected COS
> cells using the Hirt technique. Although I can get plasmid out, the
> efficiency is relatively low: less than 10^4 - 10^5 colonies out of
> perhaps 5 x 10^4 cells. From looking at others' results I have the
> impression I should be getting over 10^6, and definitely should be
> consistently over 10^5.
> I'm doing everything by the book, as far as I can see, and have tried
> fiddling with the most obvious parameters without much luck. The COS
> transfection is pretty efficient based on surface expression.
> I have spiked the lysed cell extract with plasmid and been able to
> recover most of it (i.e. spiking with 200 ng of plasmid, going through
> the Hirt extraction procedure, and transforming bacteria with an
> estimated efficiency of >10^9 colonies per ug gave me about 1 x 10^8 -
> suggesting I lost about half the plasmid: but I seem to normally be 100 -
> 1000 fold lower than I should be.)
> It seems to me that either (1) I'm not cracking the cells efficiently at
> the beginning, or am otherwise not recovering the plasmid from the cells
> efficiently, or (2) have trouble with recovering low amounts of DNA. I
> use linear polyacrylamide as my carrier, and I'm confident I'm not losing
> the pellets.
> Any suggestions? Please!
> Ian York | I find that I have,
> iayork at panix.com | as a result of this post,
> http://www.panix.com/~iayork/ | grown to hate haiku --Jennifer Mullen
It is not so suprising that the recovery of plasmid DNA is
inefficient. It seems that COS cells due to plasmid amplification (if
your plasmid contains a SV40 ori) mesh-up your plasmids. So I think
you have to live with it.
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