How to purify small PCR frg. ? Try 8% acrylamide gel.
Mon Jul 24 05:43:32 EST 1995
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How to purify PCR fragments very cleanly in the 100-200 bp range:
CRUSH ELUTION FROM ACRYLAMIDE
This method uses an 8% acrylamide non-denaturing gel, so if you do not
have an acrylamide system (such as for protein electrophoresis) it will be
be easier to start a new project outside of molecular biology, but if you
do have an acrylamide gel system (I use the BioRad MiniProtean which is
great) this is much cleaner and only slightly more time consuming than
The gel is set up as 8% non denaturing:
8.2 ml water
1.2 ml 10X TBE
2.1 ml 40% acrylamide (premade commercial)
*10 ul TEMED
* 100 ul 10% Ammonium persulfate (freshly made)
When I pour a gel, I actually double the TEMED and APS listed above, but I
pour it very fast, as the gel sets within 5 minutes that way (and faster
when the weather is hot).
The DNA is run using the same sample buffer as in agarose. The gel is run
with 1X TBE buffer. I run the Miniprotean about 20-30 minutes at 225V
70mA, but for most purposes I tend to run the bromphenol blue completely
off the gel and stop the gel when the Xylene Cyanol FF (the green dye) is
nearing the bottom of the gel. There is a table of how fast the dye front
moves on the inside cover of the "red book" Current Protocols in Molecular
Biology (the Harvard Massachusetts General manual).
A good set of low MW markers is crucial (eg 123 ladder). I recommend
the first time you try the technique, you have one lane with your markers
and another lane with only one known fragment of ~100 bp (this way you
know which band is which of your markers.)
If you have a large sample, you may need wide preparative wells or to
split your sample into several wells. If you overload a lane, the bands
will be smeary.
To stain the gel, leave the gel stuck to one of the glass plates, so
you can still handle the gel, and submerge the gel (glass plate and all)
in clean water mixed with ethidum bromide (I add 5 ul of 10 mg/ml stock to
every 100 ml water). No need to shake; let stain on bench top 25 minutes
(could be longer with a thicker gel). Get the gel off the glass plate and
onto the UV, photograph, and cut out your band.
TO GET DNA OUT OF ACRYLAMIDE
I have seen several elegant methods for getting DNA out of acrylamide, but
this method is by far the simplest and is very clean. You need two
plastic eppendorf tubes: one 500 ul tube, one 1.5 ml tube. The 500 ul
tube has its cap cut off and a sterile 19 guage syringe needle is used to
poke a hole straight through the bottom of the tube. The 500 ul tube is
seated inside the 1.5 ml tube, and the gel slice (its better if it's
small) is placed inside the smaller tube such that when they are
centrifuged, the gel slice is forced to the bottom of the 500 ul tube, and
then the gel squeezes through the needle hole of the 500 ul tube and spins
to the bottom of the 1.5 ml tube. Now the acrylamide is crushed for
maximum surface area.
I then add 100-200 ul TE (3X volume of band), vortex, and incubate
overnight at 37 degrees. Next day spin the acrylamide down, harvest the
supernatant with most of your DNA, and add another 100-200 ul of TE to the
acrylamide band, vortex, centrifuge, and harvest supernatant. Pool both
supernatants (I centrifuge once again to get out the last bits of
acrylamide) and ethanol precipitate with your favorite salt and favorite
carrier (I use Qwik Precip and NaOAC).
The DNA is now clean and ready for digestion, cloning (eg TA), or
making probes. This method can also be used to get fragments clean of
In article <3uk2mc$clk at sifon.cc.mcgill.ca>, Graham Dellaire
<popa0206 at PO-Box.McGill.CA> says:
>> p_rudolph at MORPH.SPACENET.DE (Peter Rudolph) writes:
>> Hi netters,
>> two fragments: 29 and 67bp. She separated them by gel
>> electrophoresis on a 2.5% AG.
>: How to purify the bigger fragment from the
>> gel slice with a really *high* yield?
>> Any suggestions?
>> Any help will be highly appreciated!
>The only thing I can think of Electroelution or DEAE-paper.....
>Another thing you could do is use a two vector cloning method...
>You have the vector you want to clone in say with amp R and then
>a second vector with TetR....
>You clone the fragment (96 bp) into the tet R plasmid
>cut with your restriction enzyme ligate to the othe amp R plasmid
>then select for amp R/TetR
>then cut with two enzymes one near your fragment and another neat where
>two vector sequences are ligated together such that your "DNA" is in only
>AmpR half of your plasmid then plate and select for ampR.
>If this is hard to follow (as I know it must be) there is a reference In
>while back (Jan 1995 or Dec 1995)
>Graham Dellaire Snail Mail:
> Red Cross, Research
>McGill University Montreal Blood Services
>Faculty of Medicine 3131 Sherbrooke St. East
>Div. of Experimental Medicine Montreal, QC, Canada
>E-mail: popa0206 at po-box.mcgill.ca H1W 1B2
>B2XE at musicb.mcgill.ca
>WWW Page: http://www.medcor.mcgill.ca/EXPMED/expmed.html
>Fax: (514) 525 0881
>Voice: (514) 527 1501 ext 175
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