PROTOCOL

Silver staining DNA in polyacrylamide gels

Brant J Bassam1 & Peter M Gresshoff2
1Corbett Life Science, Brisbane Technology Park, 42 McKechnie Drive, Eight Mile Plains, Queensland 4113, Australia. 2ARC Centre of Excellence for Integrative Legume
Research, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia. Correspondence should be addressed to P.M.G. (p.gresshoff@uq.edu.au).

Published online 25 October 2007; doi:10.1038/nprot.2007.330

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with
unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA
in the picogram range can be reliably detected. The described protocol is fast (B1 h) and is implemented using readily available
2007 Nature Publishing Group http://www.nature.com/natureprotocols

chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important.
The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image
contrast and less risk of staining artefacts.

INTRODUCTION
The separation of complex DNA samples and other biological
molecules with high resolution by polyacrylamide gel electrophor-
esis (PAGE) has broad application. However, to realize the potential
of PAGE, a visualization method offering superior clarity and
sensitivity is also required. The silver staining method described
here has proven very effective in this regard.
As a method, silver staining was originally developed to detect
proteins separated by PAGE13. It was further optimized and
applied to visualize other biological molecules, for example, nucleic
acids4,5, lipopolysaccharides6, glycoproteins and polysaccharides7.
These earlier protocols were, however, comparatively tedious and
offered limited sensitivity.
The development of DNA amplification fingerprinting (DAF) by
Caetano-Anolles et al.8 required a superior protocol to adequately
resolve and visualize complex DNA profiles. These requirements
led directly to the codevelopment of a successful combination of
polyester-backed PAGE gels and DNA silver staining. The silver
stain protocol developed for DAF was described separately by
Bassam et al.,9 and has since gained wide acceptance including
commercialization (e.g., in the GenePrint STR systems and SILVER
SEQUENCE products from Promega Corporation, USA).
Silver staining of DNA (and other biological samples) has several
advantages:
 Image development and visualization is done under normal
ambient light. Thus, the procedure can be performed entirely at
the laboratory bench without the need for darkroom or UV
illumination facilities.
 The image is resolved with the best possible sensitivity and detail,
because silver is deposited directly on the molecules within the
transparent gel matrix. Thus visualization is from the primary
source and does not suffer any degradation or blurring that can
accompany secondary imaging devices which involve fluores-
cence, autoradiography, focusing lenses, film development or
digital image processing.
 Silver staining offers similar sensitivity to autoradiography, but
avoids radioactive handling, delays from development times and
waste disposal issues.
 As a preferred option, gels can be dried onto a semi-rigid plastic
backing film such as GelBond PAG film, creating a permanent
record of the original material8 (see ref. 10 for a revised
protocol). Air-dried gels are resilient, preserving a concentrated
and contrast-intensified image. They can also be stored indefi-
nitely without distortion, obviating the need and added expense
of photography and printing. In addition, the preserved gel is a
molecular archive, as stained DNA bands are real DNA that
can be extracted, amplified, cloned and DNA-sequenced.
While the protocol described here focuses on the visualization of
DNA by PAGE, the method can be used without modification to
visualize a wide range of biological molecules including RNA,
polysaccharides, lipopolysaccharides, proteins and polypeptides
in polyacrylamide gels (data not shown). Although this is an
advantage in terms of scope, it nevertheless means that the protocol
must be applied with due care; stray human fingerprints on the gel
surface stain with perfect detail as will almost any other biological
impurity incorporated into or onto the gel matrix. It is thus
important to use dust-free reagents of the best analytical grade,
including the purest water available.

MATERIALS
REAGENTS . Sodium carbonate powder, Z99.5%, ACS reagent (Na2CO3; e.g.,
. Acetic acid, glacial, AR Select (ACS) (CH3COOH; e.g., Mallinckrodt, cat. no. Sigma-Aldrich, cat. no. 223530) m CRITICAL For best results, must be of
v193) m CRITICAL For best results, must be of high purity. high purity.
. Silver nitrate crystal, AR (ACS) (AgNO3; e.g., Malinckrodt, cat. no. 2169) . Ethanol
(see REAGENT SETUP) m CRITICAL For best results, must be of high . Polyacrylamide gel
purity. . Fixer solution (see REAGENT SETUP)
. Formaldehyde solution, AR (ACS) (HCHO; e.g., Malinckrodt, cat. no. 5016) . Developer solution (see REAGENT SETUP)
(see REAGENT SETUP) m CRITICAL For best results, must be of high . Developer stop solution (see REAGENT SETUP)
purity. EQUIPMENT
. Sodium thiosulfate (Na2S2O3; e.g., Sigma, cat. no. S7026) (see REAGENT . Plastic storage carboys with spigot (from any general laboratory
SETUP) m CRITICAL For best results, must be of high purity. supplier)

NATURE PROTOCOLS | VOL.2 NO.11 | 2007 | 2649

 PROTOCOL
. Laboratory gloves from any general laboratory supplier m CRITICAL
Nonpowdered.
. Platform rocker or orbital shaker (from any general laboratory supplier)
(see EQUIPMENT SETUP)
. Silver discard bottle (add NaCl to precipitate silver) (from any general
laboratory supplier)
. Discard bin from any general laboratory supplier
Optional: To preserve the original gel undistorted, we use polyester-backed
polyacrylamide mini-gels, an updated protocol for which is described by
Bassam and Bentley10
. Venturi (aspirated type) pump
. Mini-Protean II apparatus (Bio-Rad Laboratories, Hercules, CA)
. Sony digital DSC R1 camera (Sony)
REAGENT SETUP
Fixer solution Dilute glacial CH3COOH to 7.5% (vol/vol) with deionized
2007 Nature Publishing Group http://www.nature.com/natureprotocols
solution must be made up fresh as required. m CRITICAL Before preparing,
estimate the volume needed for the number and size of gels that are to be
stained.
Sodium thiosulphate stock solution Dissolve 0.2 g sodium thiosulphate in
50 ml water to make a stock solution. m CRITICAL The stock must be prepared
fresh weekly, hence keep it as small as possible to avoid wastage. m CRITICAL
Before preparing, estimate the volume needed for the number and size of gels
that are to be stained.
Developer solution Dissolve 3 g Na2CO3 in 100 ml deionized water to make
the developer solution. To speed dissolving and avoid clumping, swirl the water
vigorously and add the Na2CO3 gradually. m CRITICAL This solution must be
made up fresh as required and used at B8 1C. This is most conveniently done by
swirling the solution on an ice bath and monitoring the temperature just before
use. To raise the temperature, should it get too cold, swirl the flask under a hot
water tap. m CRITICAL Before preparing, estimate the volume needed for the

water. Store at room temperature (1825 1C). A plastic carboy with spigot makes
a suitable storage container. Fixer solution is stable and can be made up in bulk
(e.g., 4 l stock volume). ! CAUTION Solution is slightly corrosive (household
vinegar is commonly 5% CH3COOH). Avoid inhaling the vapor.
Formaldehyde solution Add 15 ml formaldehyde to 85 ml deionized water.
! CAUTION Components of this solution are toxic; handle and dispose of with
care. Consult local Occupational Health and Safety legislation. m CRITICALThis
solution must be made up fresh as required. Ensure formaldehyde is stored
at room temperature, since cold storage (i.e., 4 1C) causes inactivation.
m CRITICAL Before preparing, estimate the volume needed for the number
and size of gels that are to be stained.
Silver solution Dissolve 0.1 g AgNO3 in 100 ml deionized water. ! CAUTION
Components of this solution are toxic; handle and dispose of with care. Again,
consult local Occupational Health and Safety legislation. m CRITICAL This
number and size of gels that are to be stained.
Developer stop solution Dilute glacial CH3COOH to 7.5% (vol/vol) with
deionized water. Store refrigerated at 4 1C. A plastic carbouy with spigot
makes a suitable storage container. Solution is stable and can be made up
in bulk (e.g., 4 l stock volume). ! CAUTION Solution is slightly corrosive.
Avoid inhaling the vapor. m CRITICAL Before preparing stock solutions,
estimate the volume needed for the number and size of gels that are to
be stained.
EQUIPMENT SETUP
Platform rocker The most specialized equipment item required is the platform
rocker. A simple and gentle rocking motion (once every 23 s) gives the best
results. Orbital motion is not recommended as it does not distribute reagent
evenly across the gel surface, seemingly because reagent swirls around the
perimeter of the gel leaving the center region relatively stagnant.

PROCEDURE
Nucleic acid fixation
1| Choose a clean plastic staining tray that is larger than the gel by B2 cm on all sides. Pour sufficient fixer solution into the
tray to cover the gel to a depth of B5 mm.
2| Disassemble the PAGE rig carefully, and place the gel into the staining tray. If you are using a polyester-backed gel, place it
such that the gel side faces up in the tray.
3| Rock the staining tray continuously on a platform rocker. For typical mini-gels of B1 mm thickness, a minimum of 5 min
fixation is required, but 10 min provides optimum contrast. Longer times may be needed if thicker gels are used. This step may
continue for up to B30 min.
m CRITICAL STEP Fixation is important for stain sensitivity. Its main function is to immobilize the DNA molecules in the acrylamide
gel matrix to avoid diffusion and subsequent image blurring. It also removes and neutralizes unwanted chemicals such as urea and
buffer, which can interfere with staining.
4| While the gel is fixing, clean the gel rig and glass plates thoroughly. Reassemble the rig ready for the next gel casting. Rinse
the assembled rig with 96% ethanol to promote drying, and store the rig upright in a dust-free location ready for future use.

Prepare fresh solutions

5| While the gel is fixing, prepare sufficient developer solution (as described in REAGENT SETUP) to cover the gel in the
staining tray to a depth of B5 mm.
m CRITICAL STEP This and the solutions made up in the following steps are best prepared at this point in the protocol to ensure
freshness and for optimal time management. (The sodium thiosulphate stock and developer stop solutions should already be
pre-prepared and ready to use at this point.)
6| Add sodium thiosulphate stock solution (prepared as described in REAGENT SETUP) at the rate of 50 ml per 100 ml to the
developer solution.
7| Cool the developer solution by putting it into a 4 1C refrigerator.
8| Prepare sufficient formaldehyde solution (as described in REAGENT SETUP) to cover the gel in the staining tray to a depth of B5 mm.
9| Prepare sufficient silver solution (as described in REAGENT SETUP) to cover the gel in the staining tray to a depth of B5 mm.

Gel washing
10| Following fixation, carefully decant the solution, taking care not to damage the gel or touch the gel surface. Alternatively,
use a water-powered Venturi (aspirated type) pump to vacuum the fixer solution away and down the sink.

2650 | VOL.2 NO.11 | 2007 | NATURE PROTOCOLS

 PROTOCOL

11| To wash the gel, pour sufficient deionized water into the staining tray to cover the gel to a depth of B5 mm.
12| Rock the staining tray continuously on a platform rocker for 2 min. Longer times may be needed if gels thicker than B1 mm
are used. If the gel is washed for too long (over B20 min), then staining may be compromised, and fainter bands will result.
13| At the end of the wash, carefully decant the wash solution as described in Step 10.
14| Repeat the wash steps two times more for a total of three washes in deionized water.
m CRITICAL STEP Washing the gel is important. It removes acid and other trace substances that interfere with staining, and
provides a clear, blemish-free background to the final stain.

Formaldehyde pre-treatment
15| Add sufficient formaldehyde solution to cover the gel in the staining tray to a depth of B5 mm. Gently rock the staining
tray continuously on a platform rocker. For typical mini-gels of B1 mm thickness, a minimum of 5 min formaldehyde
2007 Nature Publishing Group http://www.nature.com/natureprotocols

pre-treatment is required while B10 min provides optimum contrast. Longer times may be needed if thicker gels are used.
This step may continue for up to B30 min. As an alternative, formaldehyde pre-treatment (Step 15) and silver impregnation
(Step 17) can be combined as per the original Bassam et al.9 protocol. See ANTICIPATED RESULTS to view an example gel
processed using this alternative.
m CRITICAL STEP Formaldehyde pre-treatment is important for stain sensitivity and maximum image contrast.
16| Following the formaldehyde pre-treatment, carefully decant the solution, taking care not to damage the gel or touch the
gel surface. Alternatively, use a water-powered Venturi pump to vacuum the fixer solution away and down the sink.

Silver impregnation
17| Add sufficient silver solution to cover the gel in the staining tray to a depth of B5 mm.
18| Gently rock the staining tray continuously on a platform rocker. For typical mini-gels of B1 mm thickness, a 20 min
impregnation time is usually optimal.
m CRITICAL STEP The recommended silver concentration cannot be reduced without affecting sensitivity and contrast. A careful
examination of silver impregnation times showed that optimal staining was achieved after B20 min. However, as little as 10 min is
sufficient for high-quality staining without significant loss of sensitivity. Impregnation times can be increased up to B60 min, but
greater than B90 min can cause severe image loss.
19| Following silver impregnation, carefully decant the solution, taking care not to damage the gel or touch the gel surface.
! CAUTION The silver solution is toxic and should be disposed of with care. Avoid spilling the solution, as it will permanently
stain most surfaces. We precipitate the silver from used silver solution with NaCl in glass Winchester bottles, and accumulate it
for recycling (it should turn milky in the bottle as the silver precipitates).

20| Briefly rinse residual silver solution from the surface of the gel by rinsing with B100 ml of deionized water for 510 s.
Do not rinse the gel longer than B15 s, as this step removes silver from the gel.

Image development
21| Check whether the developer is cold (it should be between 4 and 10 1C). Add sufficient developer solution to cover the gel
in the staining tray to a depth of B5 mm. Agitate the staining tray throughout image development so the developer solution is
not stagnant. Image development begins as soon as the developer solution is added. The developer solution is kept cold to
control the rate of image development, since development is usually too fast to control if done at temperatures above 10 1C.
Image development typically takes about 3 min depending on gel thickness, the reagents used and the temperature of the
reagents. Alternatively, you can add 600 ml of formaldehyde solution per 100 ml of final developer solution as per the original
method9. This may improve image contrast, but usually also increases the background and edge-staining artefacts. See
ANTICIPATED RESULTS to view an example gel processed using this alternative. Room temperature development is possible for
thin gels (less than B1 mm in thickness). See ANTICIPATED RESULTS to view an example gel processed using room temperature
reagent alternatives.
m CRITICAL STEP Decreasing Na2CO3 concentration below the recommended levels causes higher background staining and poor
image contrast. Poor staining can also result from the use of low quality or old (stale) reagents.

Stopping the reaction

22| Decant the developer solution carefully, avoiding damage to the gel or touching the gel surface.
23| Check whether the developer stop solution is cold (it should be stored refrigerated at 4 1C). Add sufficient developer
stop solution to cover the gel in the staining tray to a depth of B5 mm. As an alternative, developer stop solution kept at
room temperature can be used for thin gels (o1 mm in thickness). However, this alternative requires some practice as the

NATURE PROTOCOLS | VOL.2 NO.11 | 2007 | 2651

 PROTOCOL

image will continue to develop for several seconds after the developer stop solution is added. See ANTICIPATED RESULTS to view
an example gel.
24| Allow the gel to sit in developer stop solution for 510 min.
m CRITICAL STEP The developer stop solution contains 7.5% CH3COOH. Higher CH3COOH concentrations can cause image fading,
and should be avoided. Since development occurs quickly, it is best to stop the reaction as abruptly as possible to avoid accidental
overdevelopment. For this reason, the developer stop solution is chilled to 4 1C so that it acts as quickly as possiblethe low
temperature slows the kinetics of development and allows time for the acid to take effect. Knowing when to stop the reaction takes
some practice, as there is a tendency to let it go too far. A slight yelloworange background color is acceptable, as this largely
disappears with black-and-white photography and backlighting. Use a common flat-surface light box for gel viewing, gel
interpretation and gel recording by photography.
25| Decant the developer stop solution and rinse the gel with deionized water.
2007 Nature Publishing Group http://www.nature.com/natureprotocols

26| If desired, photograph the gel. Polyacrylamide gels can be hung from a corner using an alligator clip to dry overnight.
Dried gels are robust and can be safely handled. If properly stained, the image will not fade or darken and provides a
permanent record of the experiment (15-year-old gels are still in mint condition in the authors records). However, in a very dry
environment, the gel may curl and become brittle and crack. We therefore recommend storing the dried gel in a commercially
available zip-lock plastic bag.
? TROUBLESHOOTING

 TIMING
Steps 19, nucleic acid fixation and preparation of solutions: 1030 min (concurrent)
Steps 1014, gel washing: 1020 min
Steps 15 and 16, formaldehyde pre-treatment: 1020 min
Steps 1720, silver impregnation: 2090 min
Steps 21, image development: B3 min
Steps 2226, stop reaction: 510 min

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.

TABLE 1 | Troubleshooting table.

Problem Possible reason Solution
Poor image development Low-quality reagents Order AR-grade reagents

Poor image development Stale reagents Make up fresh stock as described in the protocol

Poor image development Formaldehyde solution was inactivated Order fresh formaldehyde and store properly
by cold storage

Random staining artefacts Fingerprints, improper handling, trace dust and Always wear gloves. Keep all solutions free of dust. Do
other biological materials contacting the gel surface not let the gel touch any surface until the image has
been fully developed

Shadow staining artefacts Detergent residue on glass electrophoresis plates20 Clean glass electrophoresis plates carefully with
distilled water. If necessary, pre-wash the plates
with ethanol or HCl

Surface staining artefacts Contaminants on the glass surface to which the As a precaution, always use the same side of the glass
gel is polymerized electrophoresis plates. Tip: scribe a small F into the
corner of each plate to identify the front side each use

Smear artefacts inside gel A smear zone banded across the gel, possibly Make up fresh or alternative buffer
disrupting the migration of DNA bands, is
characteristic. Usually due to incorrect electrophoresis
buffer or buffer containing contaminants

Smear artefacts inside gel Poor acrylamide polymerization Check acrylamide and TEMED are fresh

2652 | VOL.2 NO.11 | 2007 | NATURE PROTOCOLS

 PROTOCOL

TABLE 2 | Different treatments tested (af). Steps performed in each treatment are checked (tick symbol).
Alternative treatment a* b c d e f#
Combined formaldehyde + silver impregnation |
Separate formaldehyde pre-treatment 50% 10 min 100% 5 min 100% 1 min 100% 5 min 100% 10 min
Separate silver impregnation | | | | |
Formaldehyde added to developer solution |
4 1C Developer stop solution | | | | |
Room temperature image development and stop |
*Original Bassam et al.9 protocol.
#Standard revised (recommended) protocol.

ANTICIPATED RESULTS
2007 Nature Publishing Group http://www.nature.com/natureprotocols

To illustrate several possible alternatives of the silver staining method, polyester-backed PAGE mini-gels (using a Mini-Protean II
apparatus) were run as described10. Gels were loaded with sets of five lanes consisting of a DNA size marker and four lanes of
amplification products from a DAF experiment as example complex profiles.
Six replicate sets of the DNA samples were loaded into two gels (three sets in each gel separated by blank lanes) and
electrophoresed simultaneously back-to-back in the same apparatus.
The polyester-backed gels (GelBond PAG film) were then cut into replicate pieces and processed separately using alternative
staining regimes. Development of each gel was stopped when the image reached optimal image contrast (as judged by eye).
Six alternative treatments were used, as listed in Table 2. The results obtained are shown in Figure 1.
Interpretation of raw data depends on the type of experimental application requiring the separation and visualization of
nucleic acids. With molecular mapping, for example, polymorphic DNA markers scored as robust DNA bands of differing
mobility or presence may be used to establish genetic linkage between a segregating molecular marker (a band) and a
phenotype. Conversion of a dominant (absence versus presence) marker to a PCR-based co-dominant marker is accomplished
by isolation of the silver-stained DNA band, its reamplification, cloning and DNA sequencing. Silver staining of DNA may be
applied to co-dominant, PCR-based microsatellite (SSR) markers. Alternatively, DNA fingerprinting using a range of molecular
markers can be applied to small biological samples needed to distinguish range of individuals (whether plant, animal, bacterium
or fungus). Profiles can be scanned, visually or digitally, and then ordered to determine quantitative relativeness.
The ability to reisolate DNA from a dried silver-stained PAGE gel allows the archiving of forensic samples and evidence. This
contrasts with secondary photographic evidence as electronic files or printed images.

a b c d e f

Figure 1 | I DNA silver staining of polyacrylamide gel electrophoresis separated arbitrary primed DNA amplification products: The figure shows each of the
six treatments in order (af), as described in Table 2. Five different arbitrary DNA samples (a DNA size marker and four different arbitrarily chosen DNA
amplification fingerprinting8 profiles) were loaded into consecutive wells across two polyacrylamide gels, and electrophoresed concurrently in a Mini-Protean II
apparatus10. To allow a direct comparison of several alternative silver staining protocols, the polyester-backed gels were cut into replicate sections following
electrophoresis and stained separately. Images were photographed using a Sony digital DSC R1 camera in the same session with identical settings. Images were
cropped, and adjusted for color balance and brightness using Adobe Photoshop to produce the best possible match to actual gels (as judged by eye). Color
information was preserved to show the different hues produced by the stain. No other digital manipulation was done. All blemishes, scratches and dust particles
were left untouched.

NATURE PROTOCOLS | VOL.2 NO.11 | 2007 | 2653

 PROTOCOL

A valuable application of the procedure focuses on the verification of integrity and length of small nucleic acid molecules
(2030 nucleotides length). For example, we have used the procedure to check commercially supplied PCR primers using PAGE
gels of increased concentration (12.5%) and the here-described procedure. With increasing focus on other types of small nuclei
acids, such as small inhibitory and microRNA, one presumes that future applications will be numerous.
Here we present an updated and optimized DNA silver staining protocol that compares well with techniques previously
described in the literature1119.

ACKNOWLEDGMENTS We thank the Australian Research Council Centre of
Excellence grant scheme (CEO348212) and associated support for the Queensland
State Government, the UQ Strategic Fund and present and past members of the
laboratory. Ms. Johanna Hadler and Dr. Paul Scott (both CILR Brisbane) are
thanked for beta-testing the new procedure.
2007 Nature Publishing Group http://www.nature.com/natureprotocols
8. Caetano-Anolles, G., Bassam, B.J. & Gresshoff, P.M. DNA amplification
fingerprinting using very short arbitrary oligonucleotide primers. BioTechnology
(NY) 9, 553557 (1991).
9. Bassam, B.J., Caetano-Anolles, G. & Gresshoff, P.M. Fast and sensitive silver
staining of DNA in polyacrylamide gels. Anal. Biochem. 196, 8083 (1991).

10. Bassam, B.J. & Bentley, S. Electrophoresis of polyester-backed polyacrylamide

Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
1. Switzer, R.C. III, Merril, C.R. & Shifrin, S. A highly sensitive silver stain for
detecting proteins and peptides in polyacrylamide gels. Anal. Biochem. 98,
231237 (1979).
2. Merril, C.R., Dunau, M.L. & Goldman, D. A rapid sensitive silver stain for
polypeptides in polyacrylamide gels. Anal. Biochem. 110, 201207
(1981).
3. Heukeshoven, J. & Dernick, R. Simplified method for staining of proteins in
polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6,
103112 (1985).
4. Somerville, L.L. & Wang, K. The ultrasensitive silver protein stain also detects
nanograms of nucleic acids. Biochem. Biophys. Res. Commun. 102, 5358
(1981).
5. Boulikas, T. & Hancock, R. A highly sensitive technique for staining DNA and RNA
in polyacrylamide gels using silver. J. Biochem. Biophys. Methods 4, 219228
(1981).
6. Tsai, C.M. & Frasch, C.E. A sensitive silver stain for detecting lipopolysaccharides
in polyacrylamide gels. Anal. Biochem. 119, 115119 (1982).
7. Dubray, G. & Bezard, G. A highly sensitive periodic acid-silver stain for 1,2-diol
groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal.
Biochem. 119, 325329 (1982).
gels. Biotechniques 19, 568573 (1995).
11. Merril, C.R. Silver staining of proteins and DNA. Nature 343, 779780 (1990).
12. Rabilloud, T. Mechanisms of protein silver staining in polyacrylamide gels: a
10-year synthesis. Electrophoresis 10, 785794 (1990).
13. Guillemette, J.G. & Lewis, P.N. Detection of subnanogram quantities of DNA and
RNA on native and denaturing polyacrylamide and agarose gels by silver staining.
Electrophoresis 4, 9294 (1983).
14. Kolodny, G.M. An improved method for increasing the resolution and sensitivity of
silver staining of nucleic acid bands in polyacrylamide gels. Anal. Biochem. 138,
6667 (1984).
15. Beidler, J.L., Hilliard, P.R. & Rill, R.L. Ultrasensitive staining of nucleic acids with
silver. Anal. Biochem. 126, 374380 (1982).
16. Goldman, D. & Merril, C.R. Silver staining of DNA in polyacrylamide gels: linearity
and effect of fragment size. Electrophoresis 3, 2426 (1982).
17. Merril, C.R., Harrington, M. & Alley, V. A photodevelopment silver stain for the
rapid visualization of proteins separated on polyacrylamide gels. Electrophoresis
5, 289297 (1984).
18. Blum, H., Beier, H. & Gross, H.J. Improved silver staining of plant proteins, RNA
and DNA in polyacrylamide gels. Electrophoresis 8, 9399 (1987).
19. Merril, C.R., Goldman, D., Sedman, S.A. & Ebert, M.H. Ultrasensitive stain for
proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid
proteins. Science 211, 14371438 (1981).
20. Kruchinina, N.G. & Gresshoff, P.M. Detergent affects silver sequencing.
Biotechniques 17, 280282 (1994).

2654 | VOL.2 NO.11 | 2007 | NATURE PROTOCOLS