purify PCR products using Wizard Prep (Promega)

ashendel at aclcb.purdue.edu ashendel at aclcb.purdue.edu
Wed Jun 15 10:30:16 EST 1994


Some additional points concerning "freeze 'N squeeze" on this thread.

On 15 Jun 1994 13:28:49 GMT, 
Robert Solomon  Bioc   <rgs at mole.bio.cam.ac.uk> wrote:
>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:
>>> >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).
>>
>>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
>
>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

We use regular BRL agarose, with excellent results.  No absolute need for 
LMP, though the note about contaminants probably is correct if you intend 
to use all of your DNA fragment.  We cut more and throw some away, reducing 
the inhibitor/contaminant effect on the subsequent ligation reaction.  

>excise band of interest

It is important to minimize the band volume by trimming away the excess 
agarose.  (Be careful of UV burns when working on a light box.)

>place in eppendorf (whoops, No Name 1.5 mL microcentrifuge tube)
>cover with at least equal volume TE-saturated phenol

We found for this that water saturated (not buffered) phenol works better.

>freeze rapidly (I use N2, but suspect dry ice EtOH will be fine)

No need to be that rapid. Freezing can be done much more safely and 
conveniently in -70 or colder freezer.  This takes only a few minuets.

>spin 15 mins room temp in a microcentrifuge at top speed
>remove aqueous phase and do a couple of phenol/chloroform extractions

Be careful to avoid agaose chunks.  A second spin of the sup can help 
get rid of any agaose pieces.  We always do a final chloroform 
extraction to remove the last traces of phenol.  This phenol should be 
buffered.   Here is the most important tip:  We found that maximal yields 
of DNA was obtained only if the sample was concentrated by repeated butanol 
extraction to 100 ul just prior to precipitaton.  

>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.

Our yields usually are 50%, but sometimes are lower. 

>
>Have had tube breakages using this method - keep the neck of the
>microfuge out of the N2.
>
>RSol (who'll try anything until it stops working)
>

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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