Elution of DNA from agarose gels

37_1510 at uwovax.uwo.ca 37_1510 at uwovax.uwo.ca
Tue Nov 19 18:12:33 EST 1991


In article <8691 at gazette.bcm.tmc.edu>, steffen at mbir.bcm.tmc.edu (David Steffen) writes:
> rick at GENEMAN.WUSTL.EDU (Rick Wilson) writes:
>>K.R. Raj writes:
>>>I am quite tired of electroeluting DNA out of agarose. Could
>>>anyone suggest a better way other than using the geneclean kit ?
> 
>>I've had a lot of success with simple phenol extraction of melted
>>low-gelling temperature agarose.
> 
> I just want to second this, and to add that for many procedures, we
> find that we do not need to remove the agarose at all.  All of our
> ligations, sequencing, and random oligo primed labelling are now done
> in low melting point agarose directly.  Melt the agarose at 65 degrees

We have a good deal of experience cloning fragments extracted from agarose gels
by electrophoresis into a strip of DEAE cellulose paper (DE81, Whatman). The
method we use in the lab is as follows:
1) run a preparative agarose gel (0.6% to 1%, depending on the size of the
fragment of interest) containing 0.5 ug/ml ethidium bromide.
2) Transilluminate with a long wave lenght UV source (do not use short wave to
avoid nicking).
3) Make a cut parallel to the fragment just in front of it with a razor blade.
Do not cut the entire gel because the pieces will float in the tank later.
4) Insert a 3-4 mm width strip of DE 81 paper (previously autoclaved) and
continue the electrophoresis at about 50 mAmps for 5 min.
5) Take the paper into a 250 ul eppendorf tube with a small hole at the bottom,
and put this tube into a 1.5 ml eppendorf tube.
6) Wash 5 times with 100 ul of TE with brief spins between washes.
7) Transfer the small microfuge tubes to clean 1.5 ml tubes.
8) Elute the DNA with 5 times 100 ul of 1M NaCl.
9) Add 1 ml ethanol and keep at -20 for at least 30 min.
10) Spin down, wash with 70% ETOH, dry and go for it!

Some precautions:
a) If your DNA fragment is small and migrates with the dye front, use xylene
cyanol dye instead of bromophenol blue. BFB will be eluted and affect ligation.
b) Make a small hole with a heated needle. Big holes will let pieces of paper
come through. This also affects ligation.
c) Do all the steps with gloves.

We have cloned fragments of various sizes, and also used DNA fragments isolated
by this method (even a 14 Kb fragment) as templates for in vitro
transcription-translation.  By the same token, we use this method for preparing
probes.

The only expense is the paper (about $300), but there comes so much that you
buy it only once in a life time.

Miguel A. Valvano, Microbiology and Immunology, Univerisity of Western Ontario,
London, Ontario, Canada, N6A 5C1.
> reaction at the usual temperature.  (It doesn't matter if the agarose
> hardens again durring the reaction).  We have also done restriction
> digests this way, but for technical reasons it is usually easier to
> purify the reprecipitate the DNA first in this case.
> 
> BTW, people in my lab are quite superstitious about sources and lots
> of agarose.  The only LMP agarose they will use is Genetic Technology
> Grade SeaPlaque GTG agarose from FMC BioProducts.  I have no
> connection with FMC except as a satisfied customer, and I certainly
> don't mean to suggest that other brands don't work.  I just want to
> suggest that if you have difficulties, you might try a different
> source of agarose.
> 
> -David Steffen-
> -- 
> David Steffen
> Department of Cell Biology, Baylor College of Medicine, Houston TX 77030
> Telephone = (713) 798-6655, FAX = (713) 790-0545
> Internet = steffen at mbir.bcm.tmc.edu



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