purify PCR products using Wizard Prep (Promega)
lab_winoto at maillink.berkeley.edu.
Tue Jun 28 16:25:21 EST 1994
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
> normal agarose to seperate the PCR products, cut the band, and boil
> it to melt the agarose. Then I follow the Promega's steps to mix the
> DNA products with the resin, add it into the column, and elute it.
> It is said the recovery can be >90%, but we only get <10%. Are there
> any of you has ever utilized this method to purify DNA from agarose gel?
> Any suggestion will be great appreciated.
I've never used the promega kit to isolate DNA from agarose, but it
seems to me that if you boil regular agarose to melt it you would
denature your DNA. Does the agarose melt at a lower temperature with
the promega kit? I've routinely used the lithium chloride method of
DNA isolation from lowmelt agarose and its worked quite well. This
technique was described in Biotechniques but I don't remember which
issue. Briefly, resolve your DNA on regular melt agarose. After the
band your interested in has resolved, stop the gel and using a razor
cut a trough in front of the band. Remove excess buffer with a pipet
and fill the trough with lowmelt agarose containing EBr. The LMT
agarose should have a concentration of 1.0% or at least 0.1% higher
concentration than the resolving gel. After the gel has cooled run the
fragment into the LMT agarose. Cut the band out in the minimal volume
(usu<90mg). Trim away portions of the agarose that have only diffuse
concentrations of DNA, use only the rich heart of the band. Place in
eppendorf tube and add 1 volume H20. Melt at 72C for 5 minutes and
add one volume of equilibrated phenol (pH>7.6), vortex 1-2 seconds and
centrifuge for 5 minutes. Transfer the aqueous phase to a fresh tube,
don't worry about picking up a little agarose it will be removed in the
next step. Add 1/10 volume of 4M LiCl, mix and incubate on ice for 5
minutes. Centrifuge and transfer the resulting upper aqeous phase to a
fresh tube. Add 1/10 volume of 3M NaOAc pH 6.0 and 2.5 or more volumes
of EtOH. Mix and centrifuge for 20 minutes (you can chill it first if
you want). Remove the EtOH by aspiration, be careful the pellets are
very small, wash once with 80% EtOH, spin, remove EtOH, air dry briefly
and resuspend in 10 ul of H20. You should get plenty of DNA.
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