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Protocol

Isolation of DNA Fragments from Polyacrylamide Gels

by the Crush and Soak Method
Michael R. Green and Joseph Sambrook

The standard method to recover fragments of DNA from polyacrylamide gels is the “crush and soak”
technique. The eluted DNA is generally free of contaminants that inhibit enzymes or that are toxic to
transfected or microinjected cells. The method requires time but little labor and results in recovery of
<30%–90%, depending on the size of the DNA fragment. It can be used to isolate both double-
stranded and single-stranded DNAs from neutral and denaturing polyacrylamide gels, respectively.
The method is widely used to isolate synthetic oligonucleotides from denaturing polyacrylamide
gels. DNA recovered from polyacrylamide gels by crushing and soaking is generally suitable for use
as a hybridization probe, as a polymerase chain reaction (PCR) primer, and as a substrate in enzyme-
catalyzed reactions.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.

Reagents
Acrylamide gel elution buffer <R>
Other buffers may be substituted for acrylamide gel elution buffer. For example, if the DNA fragment is radiola-
beled and is to be used as a hybridization probe, hybridization buffer can be substituted.

Anion-exchange resins (e.g., QIAGEN-tips, DNAPac (Dionex); optional; see Step 12)
Carrier tRNA (optional; see Step 9)
Chloroform (optional; see Step 8)
DNA markers
Markers are commercially available or can be generated easily by restriction digests of known quantities of a well-
characterized DNA.

DNA sample
Ethanol (70%, 95%)
Gel-loading buffer (6×)
Phenol:chloroform (1:1, v/v; optional; see Step 8)
Polyacrylamide gel of the appropriate concentration with appropriate buffer

From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.
© 2019 Cold Spring Harbor Laboratory Press
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143
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M.R. Green and J. Sambrook

Polyacrylamide gel or high-resolution agarose gel of the appropriate concentration with

appropriate buffer
Sodium acetate (3 M, pH 5.2)
TE (pH 8.0) <R>

Equipment
Autoradiographic film (optional; see Step 2)
Column (disposable plastic) equipped with a frit (e.g., IsoLab Quik-Sep columns) or a syringe barrel
plugged with a Whatman GF/C filter or siliconized glass wool (optional; see Step 8)
Container with ice
Felt-tipped marker (optional; see Step 2)
Gel electrophoresis apparatus
Inoculating needle (optional; see Step 3)
Microcentrifuge
Microcentrifuge tubes or polypropylene tubes
Pasteur pipette, drawn out (optional; see Step 6)
Plastic wrap (e.g., Saran Wrap)
Rotating wheel/rotary platform in an incubator preset to 37˚C
Scalpel or razor blade
Ultraviolet lamp, handheld, long-wavelength (302 nm)
Vortex

METHOD

1. Perform polyacrylamide gel electrophoresis of the DNA sample and markers using standard
methods. Locate the DNA of interest by autoradiography or by examination of SYBR-stained
gels in long-wavelength (302 nm) UV light.
2. Use a clean sharp scalpel or razor blade to cut out the segment of the gel containing the band of
interest, keeping the size of the polyacrylamide slice as small as possible. This can be achieved by
any of the following methods:

i. While the DNA is illuminated with UV light, cut through both the gel and the Saran Wrap,
and then peel the small piece of gel containing the DNA from the Saran Wrap.
ii. Use a permanent felt-tipped marker (e.g., Sharpie pen) to outline the DNA band on the back
of the glass plate while the gel is illuminated from below with UV light. Invert the gel, remove
the Saran Wrap, and cut out the band using the marker outline as a guide.
iii. In the case of a fragment of DNA identified by autoradiography, place the exposed autora-
diographic film on the Saran Wrap and align it with the gel. Use a permanent marker to
outline the position of the desired DNA fragment on the back of the glass plate. Remove the
exposed film and Saran Wrap, and cut out the band.
Photograph or autoradiograph the gel after the bands of DNA have been excised to produce a perma-
nent record of the experiment.

3. Transfer the gel slice to a microcentrifuge tube or a polypropylene tube. Use a disposable pipette
tip or inoculating needle to crush the polyacrylamide gel against the wall of the tube.
Alternatively, use a clean scalpel or razor blade to slice the gel into thin slivers before placement in the
elution tube.
4. Calculate the approximate volume of the slice, and add 1–2 volumes of acrylamide gel elution
buffer to the microcentrifuge tube.

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Isolation of DNA from Gels by the Crush and Soak Method

5. Close the tube and incubate it at 37˚C on a rotating wheel or rotary platform.
At this temperature, small fragments of DNA (<500 bp) are eluted in 3–4 h; larger fragments take 12–16 h.

6. Centrifuge the sample at maximum speed for 1 min at 4˚C in a microcentrifuge. Transfer the
supernatant to a fresh microcentrifuge tube, being extremely careful to avoid transferring frag-
ments of polyacrylamide (a drawn-out Pasteur pipette works well).
7. Add an additional 0.5 volume of acrylamide gel elution buffer to the pellet of polyacrylamide,
vortex briefly, and centrifuge again at maximum speed for 1 min at 4˚C. Combine the
supernatants.
8. (Optional) Remove any remaining fragments of polyacrylamide by passing the supernatant
through a disposable plastic column equipped with a frit (e.g., IsoLab Quick-Sep columns) or
a syringe barrel plugged with a Whatman GF/C filter or siliconized glass wool.
The eluted DNA can be extracted with phenol:chloroform and chloroform to remove SDS, which can inhibit
subsequent enzymatic manipulation of the DNA. Precipitate the extracted DNA with ethanol as described
in Step 9, and continue with the remainder of the protocol.
9. Add 2 volumes of 95% ethanol at 4˚C to the flowthrough, and store the solution for 30 min on ice.
Recover the DNA by centrifugation at maximum speed for 10 min at 4˚C in a microcentrifuge.
Even small quantities of DNA are efficiently precipitated by ethanol in this method, perhaps because of the
presence of small amounts of polyacrylamide in the eluate (Gaillard and Strauss 1990). However, 10 g of
carrier tRNA (commercially available) can be added before precipitation, which may improve even further
the recovery of small amounts of DNA. Before adding the carrier, make sure that the presence of RNA will
not compromise subsequent reactions with the DNA.
10. Dissolve the DNA in 200 µL of TE (pH 8.0), add 25 µL of 3 M sodium acetate (pH 5.2), and again
precipitate the DNA with 2 volumes of 95% ethanol as described in Step 9.
11. Carefully rinse the pellet once with 70% ethanol, and dissolve the DNA in TE (pH 8.0) to a final
volume of 10 µL.
12. Check the amount and quality of the fragment by polyacrylamide or high-resolution agarose gel
electrophoresis.

i. Mix a small aliquot (
20 ng) of the final preparation of the fragment with 10 µL of TE (pH
8.0), and add 2 µL of the desired gel-loading buffer.
ii. Load and run a polyacrylamide or high-resolution agarose gel of the appropriate concen-
tration, using as markers restriction digests of known quantities of the original DNA. The
isolated fragment should comigrate with the correct fragment in the restriction digest.
iii. Examine the gel carefully for the presence of faint fluorescent bands that signify the presence
of contaminating species of DNA. It is often possible to estimate the amount of DNA in the
final preparation from the relative intensities of fluorescence of the fragment and the
markers.
Only rarely is further purification of the recovered DNA required. For oligonucleotides and DNAs <100
bp in length, the best option is chromatography on anion-exchange resins that are available commer-
cially (e.g., QIAGEN-tips, DNAPac [Dionex]).

RELATED INFORMATION

This protocol is a modification of the technique described by Maxam and Gilbert (1977, 1980).

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M.R. Green and J. Sambrook

RECIPES

Acrylamide Gel Elution Buffer

10 mM magnesium acetate tetrahydrate
0.5 M ammonium acetate
1 mM EDTA (pH 8.0)
0.1% (w/v) SDS (optional)
SDS improves the efficiency of recovery, most probably by blocking nonspecific adsorption
of DNA to the walls of the tube. However, SDS is tenacious and difficult to remove from the
eluted DNA, especially when purifying oligonucleotides on Sep-Pak columns. Perhaps the
best advice is to use SDS only when attempting to recover very small amounts (<20 ng) of
DNA >1 kb in size, where recovery is already inefficient and further losses may prejudice the
success of the experiment. This is not usually the case when purifying synthetic oligonucle-
otides, which are always relatively small and usually available in abundance.
Other buffers may be substituted for acrylamide gel elution buffer. For example, if the DNA
fragment is radiolabeled and is to be used as a hybridization probe, hybridization buffer can be
substituted.

TE Buffer

Reagent Quantity (for 100 mL) Final concentration

EDTA (0.5 M, pH 8.0) 0.2 mL 1 mM
Tris-Cl (1 M, pH 8.0) 1 mL 10 mM
H2O to 100 mL

REFERENCES

Gaillard C, Strauss F. 1990. Ethanol precipitation of DNA with linear poly- Maxam AM, Gilbert W. 1980. Sequencing end-labeled DNA with base-spe-
acrylamide as carrier. Nucleic Acids Res 18: 378–383. cific chemical cleavages. Methods Enzymol 65: 499–560.
Maxam AM, Gilbert W. 1977. A new method for sequencing DNA. Proc Natl
Acad Sci 74: 560–564.

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Cold Spring Harbor Laboratory Press

Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak
Method
Michael R. Green and Joseph Sambrook

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot100479

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