Wizard preps for "advanced" uses
Chia Jin Ngee
mcblab47 at leonis.nus.sg
Fri Jul 19 22:19:55 EST 1996
Peter Barrett (pbarrett at MBCRR.HARVARD.EDU) wrote:
: Dear Fellow Netters,
: Although I realize that there's a lot of info already out there about this, I
: was wondering if we could rehash an old topic.
: Namely, I have been wondering about "advanced" uses for the Wizard
: (Promega)/"Merlin" preps. Specifically, has anyone out there used this DNA for
: transfection or injection? Is this stuff clean enough for these kinds of
: purposes, where ordinarily one might want to use something like Qiagen or
: double-CsCl purification in order to get "super-clean" DNA? (I have used the
: Promega kit with mixed results-- some DNA's get A260/280's about 1.8, others
: about 1.6).
I have never used Celite/diatomaceous earth purification techniques for
transfection purposes. Just too risky. However, I have modified most of
the techniques/solutions found in the net and have given rise to
multipurpose solutions for DNA purification from different sources. From
plasmid preps to gel elution. All solutions used are standard in my lab
now. If anyone is interested, I will post the formulations/protocols.
One thing to note though, is that for good clean DNA, centrifugation is
better than vacuum-bed methods or column methods. As Bruce Roe has
pointed out, DNA going thru a column as a tendency to be sheared.
: On a related note, what have people's experiences been with regard to using
: these preps for cosmids? I have tried using the preps for cosmids at least once,
: and although I got a very nice yield, when I try to digest I get poor cutting:
: some bands appear but in the background of a smear (yet the undigested material
: looks great). I'm wondering if the poor digestion is the result of leftover
: EtOH in the prep, or something else (see below; I tend to think the latter
: because even after Ph/CHCl, they still don't cut properly).
Have not tried with cosmids yet so I can't give much info.
: I am also wondering what, if anything, people have noticed with regard storage
: and freeze/thaws of these preps. Several preps I've made by the Wizard method
: show a large amorphous precipitate after freeze/thawing at -20oC and I'm
: wondering if this is resin getting through the column-- tends to happen more
: with large (> 10 kb) rather than small plasmids. These preps often tend to cut
: poorly after the freeze/thaw and you can't get rid of the precipitate-- if you
: spin down, suck off the supe, then re-ppt, you get more of the stuff coming out
: of solution and spinning down. Likewise, Ph/CHCl doesn't get rid of it either.
: I asked Promega about it and they think it's a defect in the columns... but I'm
: curious to know if anyone has seen anything like this as well.
Could be leftover GuHCl or small bits of Celite/earth though I've never
experienced this with my home-made resin methods. The exact same thing
I've experienced with Qiagen preps. Didn't have time to analyse the nature
of these precipitates. I've always assumed it to be junk and have spun
them down always and taken the supernatant.
Jin Ngee, Chia
(Genie, the OligoMan)
mcblab47 at leonis.nus.sg
Views and opinions expressed are solely mine and not of my employer's.
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