Ligation

[ChenHA]

Deitiker, Philip R wrote:

> 
> Somewhat new problem. 
> I am using Promega's Midiprep kit, it appears even after attempts
> to dry off the ethanol the resulting DNA has EtOH, or at least, does not freeze and has the trace smell of Ethanol. Last time I did an EtOH
> precipitation on the midiprep DNA, problem is that much of the precipitated material did not resolubilize, possibly because I incubated it too long at 65'C I am weary of doing this again so we were really careful to dry the membrane, but I still recovered a great amount of DNA. Has anyone used the
> Promega Midiprep kit and had similar problems?
> 
> Also, apparently the tube cultures of bacteria seem to work fine, but a similar volume of flask culture seems to overwhelm the kit. Too much
> DNA? Is it possible to clog the DNA binding membrane with too much DNA?

I haven't use the Promega kit for a long time, but I think most kits 
work on the same principle.  I normally use Qiagen midiprep kit, 
although other companies do kits which work well.  I am puzzled as to 
why ethanol contamination should be a problem, since you should be able 
to see if it is properly dried - if you can't see any liquid, then 
whatever ethanol that's left should be so small that it shouldn't affect 
most things you do. I normally just pipette the liquid out, leave it for 
a minute or two at room temperature, then pipette the remaining liquid 
again with a fine bore tip, (repeat a minute or two later if necessary 
if you can still see liquid in there), then resuspend the DNA in 50-70ul 
TE buffer after 5-10 minutes).  When the DNA is too dry, it makes it 
more difficult to resuspend.  But if you get lots of dissolved DNA 
anyway, this is entirely academic, just spin down whatever that didn't 
dissolve.

Wasn't there some issues with Promega kit? I think it's something about 
patents, so they had to change their kit components which didn't work 
well, but that was many years ago, I'd assume they fix whatever problems 
they had. (Who had the patent for the DNA prep and has the patent expired?)

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