purification of small DNA fragment
tfitzwater at somalogic.com
Wed Mar 27 11:17:04 EST 2002
>Hope someone out there can come up with something. I have a small fragment
>(~36bp) I want to digest out of a plasmid and recover for subcloning
>purposes. Tried a real thick (2.2%) agarose gel but the band was never
>visible. So I guess I could go with a TBE polyacrylamide gel and extract
>from that. However, I don't have a kit / protocol to do that. Any ideas?
>Thanks in advance for any ideas.
> Marc Busch
In my hands, oligonucleotides > 40 nucleotides long are purified by gel
electrophoresis on a denaturing 8% acrylamide, 8 M urea, 1x TBE slab gel
with wells that are 0.75 mm thick and 1 inch wide. Primers and truncated
ligands < 40 nucleotides in length are purified by HPLC if possible,
otherwise use a denaturing 12% gels. Double-stranded DNA fragments should
be isolated on nondenaturing gels.
SYBR Gold is the most sensitive gel stain for cutting out bands. Gels
should be hand stained to avoid DNA contamination from heavily used staining
baths. Prepare a 10 mL staining solution and spread it over the gel.
Alternatively, radiolabel the DNA fragment.
Prepare Elution Solution: 0.3 M sodium acetate, pH 7.5-7.8, 2 mM Na2EDTA.
Prepare 3 M sodium acetate stock by dissolving 24.612 g of anhydrous sodium
acetate with 80 mL of Type I water. Adjust the pH to 7.5-7.8 with 10N NaOH.
Prepare elution solution by combining 10 mL of 3 M sodium acetate, pH
7.5-7.8 and 400 µL of 0.5 M EDTA with 89.6 mL of Type I water. Autoclave or
filter sterilize. (Note: the pH used in this method was designed for 2'
fluoropyrimidine RNA. Normal DNA and RNA can be eluted in standard sodium
Passive elution method: transfer the gel pieces with the back of the blade
in 5 mm x 10 mm pieces to siliconized microfuge tubes (or Falcon tubes for
preparative runs) containing Elution Solution. 0.75 mm thick bands from ½
inch wells elute in 330 µL and bands from 1 inch wells elute in 1 ml of
Elution Solution with 94% and 85% efficiency, respectively.
Oligonucleotides are passively eluted at least 4 hours (usually overnight)
on a room temperature rotator. After elution, the tubes are spun briefly in
a microfuge, the aqueous contents are carefully pipetted off and ethanol
precipitated as described below. If the gel slice fragmented during
elution, transfer the sample to a Spin-X cartridge and spin briefly in a
picofuge as described below. Passive elution works for 6-15% acrylamide
gels. Bands from higher concentration gels should be extracted by a
Crush/soak method: the gel slices are placed in microfuge tubes, frozen in
dry ice-ethanol for 5 minutes and thawed (optional), 200 µL of Elution
Solution is added, and the gel slice is crushed with a microfuge tube pestle
(Kontes, Vineland, NJ) or with the plunger from a 1 mL disposable syringe
until a fine slurry is formed. Care must be taken to avoid splashing
during this procedure, especially if the fragment is radiolabeled. The
sample is diluted with an additional 400 µL of elution solution, the gel
slurry is spun through a 0.45-µM sterile cellulose acetate Spin-X filter or
pushed through a 25 mm diameter cellulose acetate syringe filter (Millipore,
Bedford, MA) to remove gel particles. The slurry may be rinsed with a small
volume of elution solution.
The eluted material is then ethanol precipitated. Linear polyacrylamide is
used as a carrier. The yields from passive elution, crush/soak, and
electroelution are identical.
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