Freeze and Squeeze
Ann M. Yezerski
Does anyone have the standard protocol for "Freeze and Squeeze" to remove
DNA from an agarose gel? If so, e-mail me at ayez...@moose.uvm.edu.
Thanks in advance!
Ann Yezerski
Glen Shearer
Ann,
We do two methods of freeze/squeeze.
1. Stain gel & cut out band --> freeze agarose cut-out in -20C freezer
for about 30 min or in
-70 freezer for about 5 min.--> put frozen chunk on a square of
Parafilm and fold Parafilm around the
agarose--> squeeze firmly with fingers and a few drops of liquid will
dribble out the crease of the parafilm
into a waiting microfuge tube. We often use this liquid directly for
random primer labeling or add sodium
acetate (final of 0.3 M) and ethanol ppt. before use.
2. The second method is to stain and cut out band and put it in a small
microfuge tube. Freeze the whole tube
as above--> remove from freezer and spin in microfuge on high speed-->
the agarose will be 'crumbly'
and will form a loose pellet--> carefully remove the aqueous soup that
has your DNA in it--> use directly
or ethanol ppt. as above.
Both methods work quickly and generally yield 50% to 75% in our lab.
Let me know how it works for you.
Glen
In article <4p9vqk$g...@swen.emba.uvm.edu>, ayez...@gnu.uvm.edu (Ann M.
Lillian Markind Bloch
Ann M. Yezerski wrote:
>
> Does anyone have the standard protocol for "Freeze and Squeeze" to remove
> DNA from an agarose gel? If so, e-mail me at ayez...@moose.uvm.edu.
cut out band from gel
put in eppendorf in freezer
allow to freeze
put frozen agarose chunk on piece of weighing paper
push down on chunk with the paper and collect all juice in pipet.
voila!
Kevin Simcox
> Ann M. Yezerski wrote:
> >
> > Does anyone have the standard protocol for "Freeze and Squeeze" to remove
> > DNA from an agarose gel? If so, e-mail me at ayez...@moose.uvm.edu.
>
Take a 500 ul microfuge tube and make a small puncture in the bottom
using a small gauge syringe needle. Place a small amount of
silconized glass wool in the bottom. Cut out the band of interest and
place into the 500 ul microfuge tube. Then place the 500 ul microfuge
tube into a 1.5 ml microfuge tube. Freeze at -70 C for 20 min. Spin for
10 min and collect liquid containing DNA fragment. The DNA fragment can
be equilibrated in a 0.3 M NaOAc, 0.01 M MgCl2 solution prior to loading
the microfuge tube. Just add 2.5 vol of ethanol after centrifugation to
ppt the DNA fragment.
Kevin Simcox
simc...@osu.edu
Robert Horton
Kevin Simcox (simc...@osu.edu) wrote:
REFERENCE:
Tautz, D. and Renz, M. (1983) Analytical Biochemistry 132:14.
---
Robert M. Horton
http://134.84.47.3 Have a :) day
"Scotty, try flushing the radioactive waste into the ventilation system!"
D. KIM
I have been using (expensive) microfuge filter cups (e.g. S&S Ultra-Free
MC). These cups cost about US$1 each, but we had a bag of them, and
noone was using them in the lab. I would take a bit of the gel slice
(this is a SMALL piece of gel) and freeze in the -80 (I am impatient).
I'd then put the frozen gel into the cup and let thaw, then spin out the
liquid in the microfuge (a 5 second pulse). I use the eluted liquid
directly for ligations.
I have since run out of the cups, and cannot justify purchasing more. I
have instead tried placing the gel slice (frozen) into the top of an
"aerosol barrier" pipet tip, and place the tip into a 1.5 ml tube. Put
the tube into a microfuge and spin for a 3 - 5 second pulse. The barrier
effectively prevents bits of agarose from passing through, and the liquid
seems to be fine for ligation. These barrier tips are much cheaper than
Ultra-Free cups.
I may try using barrier tips for other filtration needs. Maybe I can
make a micro spin-column out of one . . .
Daniel Kim
Christoph Grunau
> Does anyone have the standard protocol for "Freeze and Squeeze" to remove
> DNA from an agarose gel? If so, e-mail me at ayez...@moose.uvm.edu.
> Thanks in advance!
>
> Ann Yezerski
* Buy "amicon micropure separators" or ask amicon for a free sample
* cut out the band of interest
* put it into the separator tube
* centrifuge at max. speed for 10 min
* for ligation PhOH/CHCl3 extract and precipitate, for sequencing use directly
(The principle is the same as described in the other postings i.e. tube
with hole in the tip. But the amicon works more efficient.)
Good luck - Christoph
MacFS User
> Does anyone have the standard protocol for "Freeze and Squeeze" to remove
> DNA from an agarose gel? If so, e-mail me at ayez...@moose.uvm.edu.
> Thanks in advance!
>
> Ann Yezerski
Look at the reference by Quan and Wilkinson (1991), Biotechniques, vol.
10, p738. I use this method routinely with no problems.
Alan.
--
al...@le.ac.uk
Dept. Microbiology and Immunology
University of Leicester
LE1 9HN
James Graham
The Quan and Wilkinson technique mentioned is the key to highly
reliable consistent subcloning when both vector and insert are
so purified. All other methods of purifiying DNA from agarose, while
perhaps more aesthetically attractive, invariable result in an
occasional unacceptable degree of sample loss. In the Q&W procedure,
any two appropriate EthBr stained slices representing vector and insert
may be merely frozen for 15 minutes and spun. 3 ul of the vector slice
supernatant may then be ligated to 0, 1, and 4 ul of insert slice
supernatant in a final volume of 10 ul using a ligase buffer with PEG
(eg. BRL's).
If one has frozen competent cells tested and stocked (use the Inoue
low temperature grown Ca-Mn-Pipes method in N2) then if you can see
the two bands on a gel, you will obtain your clones, EVERY SINGLE TIME.
I often suggest this regime, and inevitably attentions are caught
by some organic extraction, glass-binding or ion exchange matrix or
other and progress slows down to a hit or miss speed. The beauty of Q&A
is there is absolutely nowhere that your sample to go - only one tube is
used.
(Dilution prior to freezing is not necessary with low-melting agarose.
Long Wave UV (300+ nm) MUST be used to visualize bands. CAUTION: DNA
so isolated cannot be precipitated from these supernatants efficiently
without a butanol extraction and the use of glycogen as co-precipitant.)
> Quan and Wilkinson (1991), Biotechniques, vol. 10, p738.
Jim
J. Graham PhD
Biology Department
Washington University of St. Louis
gerry oneill
hi
re freeze and squeeze
either buy "Spinex" tubes from eg Costar ( they are eppis with an
acetate insert)
or make your own with a 1.5, filter paper & 0.5 ml eppis with a "pin
hole" bored in the bottom
Paul N Hengen
James Graham (gra...@biodec.wustl.edu) wrote:
> Quan and Wilkinson (1991), Biotechniques, vol. 10, p738.
I'm not meaning to give you a hard time, but I just would like
to give the reference as:
@article{Qian.Wilkinson1991,
author = "L. Qian
and M. Wilkinson",
title = "{DNA} fragment purification: removal of
agarose 10 minutes after electrophoresis",
journal = "BioTechniques",
volume = "10",
number = "6",
pages = "736-738",
month = "jun",
comment = "recovery of DNA from low temperature agarose",
year = "1991"}
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Paul N Hengen
D. KIM (dk...@nmsu.edu) wrote:
: I may try using barrier tips for other filtration needs. Maybe I can
: make a micro spin-column out of one . . .
: Daniel Kim
Sure can! I've used cut-off ART tips for plasmid mini-preps instead of using
those expensive frit thingys you get with kits. I'm convinced the huge cost of
mini-prep kits is due to the different plastic do-dads they give you to recover
the DNA from the binding matrix :-)