(by hzhen from freeuk.com)
Fri Jun 29 20:10:05 EST 2007
Deitiker, Philip R wrote:
> My helper was concerned because the membrane clogged up on
> one sample and we were forced to discard some of the lysate.
I don't know much about the Promega kit, so what I say may be
irrelevant, but things can get clogged up by, say, precipated cell
debris that wasn't properly removed, or cells not lysed and precipitated
properly (can happens if too large a volume of cells are used, so just
use proportionally more reagents in this case).
> Typically I am retreaving 90ng/ul of DNA.
> The vector is 6300 bp so this should give about 2.5 fm/ul
> [Who wanted fremptomoles? :^) ]
Seems good enough. You don't need a huge amount of DNA for ligation, in
fact, too high a concentration of DNA is bad for ligation - they tend to
form concatemers rather than circularised plasmids.
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