purify PCR products using Wizard Prep (Promega)

[Robert Solomon Bioc]

In article <dbradley-150694115340 at gen23.gen.tcd.ie> dbradley at vax1.tcd.ie (Dan Bradley) writes:
>In article <2tmf3v$4ma at lyra.csx.cam.ac.uk>, rgs at mole.bio.cam.ac.uk (Robert
>Solomon (Bioc)) wrote:
>
>> In article <CrE9Gu.Cx7 at acsu.buffalo.edu> vp23qw83 at ubvms.cc.buffalo.edu (Xin Guo) writes:
>> >Hi, Bionetters:
>> >
>> >I have a trouble to get a high yield of PCR products by using Promega's
>> >Wizard Prep method. Instead of using lower melting agrose gel, we use

<rest of Xin Guo's woes deleted - if only life were so simple>

>> known as ...  The Promega Effect  ... <cue sinister music>
>> 
>> There seems to be batch-dependent (in)efficiency in Wizard PCR Preps,
>> or some variable not taken into account by the protocol - use one
>> of the glass milk methods (no, no vested interest here, just bitter
>> experience - standard disclaimer applies) or freeze-squeeze (yes, I'd
>> rather use phenol and purify some DNA than use a kit and lose it).
>> 
>> Cheers
>> 
>> RSol
>  
>
>
>As a fellow victim of the Promega Effect - I am interested to find out what
>exactly "freeze squeeze" is and how good a purification technique it is. 
>Phenol would definitely be the lesser of two evils.
>
>Thanks
>-- 
>Dan Bradley
>EMAIL dbradley at vax1.tcd.ie

Not sure of the origins of freeze-squeeze, but as practised by me it
goes something like this <cue classy jazz piano>...

Run lmp-agarose gel
excise band of interest
place in eppendorf (whoops, No Name 1.5 mL microcentrifuge tube)
cover with at least equal volume TE-saturated phenol
freeze rapidly (I use N2, but suspect dry ice EtOH will be fine)
spin 15 mins room temp in a microcentrifuge at top speed
remove aqueous phase and do a couple of phenol/chloroform extractions
NH4Ac/i-PrOH ppte etc...

DNA is clean of contaminating DNA, RNA, protein, and if you use the 
better lmp-agaroses available, then anything left doesn't interfere
with subsequent uses, e.g. ligations.  260/280 is usually around 1.4 - 1.5,
which isn't great, but it seems to work, and you will end up with some
DNA, which isn't always the case with some purification methods ;-)
I don't know what percentage of my DNA I'm getting back - enough to
work with.

Have had tube breakages using this method - keep the neck of the
microfuge out of the N2.

Good luck

RSol (who'll try anything until it stops working)