Recovery of DNA from
Agarose Gels

Section VI: Recovery of DNA from Agarose Gels

In This Section
Tips for Increasing DNA Recovery Efficiency
from Agarose Gels
b-Agarose Recovery of DNA from Agarose Gels
Electroelution of DNA from Agarose Gels
Phenol/Chloroform Extraction of DNA from
Agarose Gels
"Modified Freeze/Squeeze" Extraction of DNA from
Agarose Gels
Ethanol Precipitation of DNA Recovered from
Agarose Gels
References

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Section VI: Recovery of DNA from Agarose Gels
Tips for Increasing DNA Recovery Efficiency from Agarose Gels

Introduction

Buffer types

This section discusses various tips which will increase
the efficiency of recovery of DNA from agarose gels. These
tips and recommendations can be applied to all recovery
techniques.

When recovering DNA from agarose gels, 1X Tris-acetate(TAE) Buffer is recommended for electrophoresis.
Casting and DNA loading tips—— Cast a gel 3 mm - 4 mm thick—— Gel casting and DNA loading tips—— Use a combs ≤1 mm thick—— Staining and recovery tips—— Load no more than 100 ng of DNA per band

Choosing the appropriate agarose for recovery

Staining and recovery tips

When recovering DNA, the choice of agarose is one of themost important factors. To avoid recovery altogether, one
can choose to perform in-gel reactions.

When recovering DNA from agarose gels, we recommend
the following:—— Stain the gel for 15 - 20 minutes

Lonza offers Genetic Technology Grade™ (GTG®) Productsthat are specially prepared for demanding molecularbiology applications. Lonza’s GTG® Product quality tests gobeyond standard nuclease assays to include enzymatic
performance measurements. Our additional testing
provides a more realistic index of overall product quality
and reliability. You no longer need to screen agarose lots
to find those which yield biologically active DNA.

—— Destain the gel in distilled water for two, 20-minutewashesLonza agaroses and compatible recovery techniques—— Cut the smallest gel slice possible■

SeaPlaque® Agarose■

MetaPhor® Agarose

NuSieve® GTG® Agarose—— Choosing the appropriate electrophoresis buffer

SeaPlaque® GTG® Agarose®

—— Prepare the gel in 1X TAE Buffer

SeaKem® GTG® Agarose

Recovery of DNA from
Agarose Gels

The section is divided into the following topics:—— Choosing the appropriate agarose■■

Phenol/ Chloroform

■

■

■

■

■

Recovery Columns

■

■

■

■

■

Electroelution

■

■

■

■

■

Freeze/ Squeeze

■

■

■

■

■

Cat. No.
In-Gelb-Agarase■—— Do not cast the gel with ethidium bromide—— Do not expose the DNA to UV light for any longer than
1 minute; long exposure of DNA to UV light can nickthe DNA—— The addition of 1 mM guanosine or cytidine to the gel
and electrophoresis buffer is effective in protecting
DNA against UV-induced damage
It is possible to avoid staining samples which will be used
for recovery by running an additional lane containing asmall amount of your sample immediately next to themolecular weight marker. However, DNA is damaged by
UV light in the absence of ethidium bromide so keep
exposure to UV light as brief as possible. Cut the lanes
containing the marker and the small amount of thesample from the rest of the gel and stain. To recover the
preparative loading, line up the stained portion of thegel with the unstained portion. Check by placing on UVtransilluminator and cut out the area that lines up withyour sample on the unstained portion of the gel.
The FlashGel® System for Recovery, (page 20) offers a
non-UV alternative for DNA recovery.

Reference
Grundemann, D. and Schomig, E., BioTechniques 21(5): 898 - 903, 1996.

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Section VI: Recovery of DNA from Agarose Gels
ß-Agarase Recovery of DNA from Agarose Gels

Introduction
ß-Agarase is an enzyme that digests thepolysaccharide backbone of agarose to alcohol-soluble
oligosaccharides. DNA in a low melting temperature
agarose gel can be recovered after the gel is melted
and digested with this enzyme. The remaining
oligosaccharides will not gel or interfere withsubsequent DNA manipulations such as cloning,labeling, restriction digestion or sequencing. Lonza’s
ß-Agarase is free of any detectable DNase, RNase
and phosphatase activities.
ß-Agarase recovery is particularly useful for recoveringlarge DNA (>10 kb) which could be sheared by othermethods of recovery.
Compatible agaroses—— SeaPlaque® GTG® Agarose (certified and tested for therecovery of DNA)—— NuSieve® GTG® Agarose (certified and tested for therecovery of DNA)—— SeaPlaque® Agarose
Tips—— Transfer no more than 200 mg of the agarose gel toa microcentrifuge tube for equilibration with digestion
buffer—— For efficient recovery of small nucleic acids (<500bp) and/or very dilute samples (<0.05 µg/ml), we
recommend either carrier tRNA or nuclease-freeglycogen be added; in addition, overnight precipitation
at room temperature can be helpful—— The enzyme preparation has maximum activity
between pH 6 - 7 and is relatively unaffected bysalt concentrations between 0.1 M and 0.25 M; theequilibration of the gel slice with ß-Agarase Buffer isnecessary to provide the enzyme with optimal bufferconditions; this equilibration is more important forgels prepared in TBE than in TAE Buffer due to thegreater buffering capacity of TBE Buffer

Recovery of DNA from
Agarose Gels

Returnto Main—— Ethanol precipitate the DNA at room temperature oron ice. The most effective DNA precipitation can beachieved at 0°C to 22°C, rather than at -20°C to -70°C.
At higher temperatures, yields are more consistentand precipitation of oligosaccharides will be avoided
ß-Agarase unit definition—— One unit of Lonza ß-Agarase will completely digest
200 mg of a molten 1% SeaPlaque® GTG® Agarose gel
which has been dialyzed in 1X ß-Agarase Buffer in 1
hour at 40°C; similar activities are obtained for otherlow melting temperature agaroses such as NuSieve®
GTG® Agarose.
—— Concentration: 1 unit per µl—— Completely melt the gel slice prior to the addition of
enzyme—— Ethanol precipitations should be incubated at room
temperature with ammonium acetate rather thansodium acetate to decrease the likelihood of coprecipitation of agarose-oligosaccharides with theDNA—— Polynucleotide kinase is inhibited by ≥7 mM
ammonium ion; use sodium acetate for yourprecipitation if you will be phosphorylating your DNA
after recovery—— The enzyme preparation retains full activity for several
hours at 45°C; however, it will gradually lose activity
upon longer incubations; for this reason, incubate at
40°C for overnight digestions

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Section VI: Recovery of DNA from Agarose Gels
ß-Agarase Recovery of DNA from Agarose Gels — continued

Recovery of DNA from
Agarose Gels

Procedure for ß-Agarase digestion

12.

Mix.

B efore you begin: Set one water bath or heating block for
70°C. Set another water bath or heating block for 45°C.

13.

Incubate at 45°C for 60 minutes.

14.

If the DNA is ≤30 kb...
Place the solution at -20°C for 15 minutes. Centrifuge
the solution for 15 minutes at 4°C.
Continue with Step 15
OR
The sample can be concentrated without ethanol
precipitation by centrifugation in a molecular weightcut-off-spin column (e.g., Amicon®’s Microcon™ Microconcentrator).
If the DNA is >30 kb...
The DNA/ß-Agarase solution may be used directly
without precipitation for subsequent enzymatic
manipulations.
OR
The sample can be concentrated without ethanol
precipitation by centrifugation in a molecular weightcut-off-spin column (e.g., Amicon’s Microcon orCentricon™ Microconcentrators).

15.

Transfer the supernatant to a new microcentrifuge tube
leaving behind any undigested agarose in the pellet.

1.

Electrophorese DNA in a low melting temperature agarosegel (such as SeaPlaque®, SeaPlaque® GTG®, MetaPhor®
or NuSieve® GTG® Agarose) which has been prepared in
TAE or TBE Buffer.

2.

Briefly stain the gel with GelStar® or SYBR® Green I
Nucleic Acid Stain or ethidium bromide.

3.

Photograph the gel.

4.

Excise a gel slice containing the DNA of interest.

5.

Place the gel slice containing the DNA sample into a
microcentrifuge tube.

6.

Equilibrate the gel slice with 10 volumes of 1X ß-Agarase
Buffer for 60 minutes at room temperature OR follow
steps 6A - 6D.
6a.

Melt the gel slice at 70°C for 15 minutes. Make
sure that the gel slice is completely melted as
the enzyme preparation will not digest agarosein the gelled state.

6b.

Add 2 µl of the 50X ß-Agarase buffer toapproximately 100 µl of melted gel solution.

16.

Add ammonium acetate to the supernatant to a final
concentration of 2.5 M.

6c.

Mix the solution.

17.

Add 2 to 3 volumes of 100% ethanol.

6d.

Proceed to steps 9, 10 and 11.

18.

If the DNA is ≤0.05 µg/ml. . .

7.

Discard the buffer.

8.

Melt the gel slice at 70°C (approximately 15 minutes).
Make sure the gel slice is completely melted as the
enzyme preparation will not digest agarose in the gelledstate.

9.

Cool the melted agarose solution to 45°C.

10.

Add 1 unit of ß-Agarase for 200 mg (approximately

200 µl) of 1% agarose gel (add proportionally more orless of the enzyme preparation for larger or smaller gel
slices, or with higher or lower agarose concentrations,
respectively). Skip to step 12.
11.

Precipitate at room temperature for 24 hours
OR
Add 10 µg of RNA.
If the DNA is >0.05 µg/ml. . .
Precipitate at room temperature for 30 minutes.
19.

Collect the precipitate by centrifugation.

Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

If you have followed the protocol in steps 6A - 6D, theamount of enzyme will need to be adjusted.
If the gel was
prepared with . . .

Then you willneed to add . . .

TAE Buffer
TBE Buffer

Twice the amount of enzyme
Seven times the amount of enzyme

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Section VI: Recovery of DNA from Agarose Gels
ß-Agarase Recovery of DNA from Agarose Gels — continued■■ Materials–– Microcentrifuge tubes (≥1 ml)–– Two water baths or heating blocks–– Scalpel or razor blade–– Microcentrifuge■■ Reagents

Recovery of DNA from
Agarose Gels

Returnto Main–– SeaPlaque®, SeaPlaque® GTG®, NuSieve® GTG® or
MetaPhor® Agarose–– GelStar® or SYBR® Green I Nucleic Acid Gel Stain or
ethidium bromide–– ß-Agarase buffer–– 	Supplied as a 50X concentrate; 1X concentration is:
40 mM Bis Tris/HCl 40 mM NaCl, 1 mM EDTA (pH
6.0)–– ß-Agarase Enzyme–– 10 M ammonium acetate–– 100% ethanol
Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

References
Chong, S. and Garcia, G.A., BioTechniques 17: 719 - 725, 1994.
Crouse, J. and Amorese, D., gibco-BRL Focus 9: 3 - 5, 1987.
Lamb, B.T., et al., Nature Genetics 5: 22 - 29, 1993.
Morrice, L.M., et al., Can. J. Microbiol. 3: 987 - 933, 1983.
Richardson, C.C., The Enzymes, 3rd Edition, Academic Press, 1981.
Zeugin, J.A. and Hartley, J.L., gibco-BRL Focus 7(4): 1 - 2, 1985.

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Section VI: Recovery of DNA from Agarose Gels
Electroelution of DNA from Agarose Gels

Introduction■■ Materials–– Scalpel or razor blade

Recovery of DNA from
Agarose Gels

Electroelution is a reliable and consistent recovery
method. The recovered DNA is suitable for a wide range
of applications. For small fragments, a typical yield would
be 50 - 85%, but as fragment length increases, yieldcan drop as low as 20%. The procedure below describes
the electroelution of DNA into dialysis bags and has
been adapted from Sambrook, et al. Alternatively, if you
have a commercially available apparatus, follow the
manufacturer’s instructions.
Compatible agaroses–– Spatula–– Dialysis tubing–– Dialysis tubing clips–– Electrophoresis chamber–– Hand-held, long-wavelength ultraviolet lamp–– Disposable plastic tube–– Pasteur pipette■■ Reagents—— SeaKem® GTG® Agarose (certified and tested for therecovery of DNA)–– 1X TAE Buffer–– 2% w/v sodium bicarbonate, 1 mM EDTA
pH 8.0, prepare in distilled water—— SeaPlaque® GTG® Agarose (certified and tested for therecovery of DNA)–– 1mM EDTA, pH 8.0—— NuSieve® GTG® Agarose (certified and tested for therecovery of DNA)–– Distilled water–– GelStar® or SYBR® Green I Nucleic
Acid Gel Stain or ethidium bromide—— SeaPlaque® Agarose—— NuSieve® 3:1 Agarose

Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

—— MetaPhor® Agarose
Tips–– Electrophorese DNA in 1X TAE Buffer–– Have 1 µg of DNA in your band of interest–– Minimize exposure of DNA to UV light for no morethan 1 minute–– Cut the smallest gel slice possible

Procedure for preparing dialysis tubing
1.

Cut the tubing into 10 cm to 20 cm long pieces.

2.

Boil for 10 minutes in a large volume of 2% sodium
bicarbonate/1 mM EDTA, pH 8.0.

3.

Rinse the tubing with distilled water.

4.

Boil for 10 minutes in 1 mM EDTA, pH 8.0.

5.

Cool the tubing.

6.

Store at 4°C submerged in 1 mM EDTA, pH 8.0.

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Section VI: Recovery of DNA from Agarose Gels
Electroelution of DNA from Agarose Gels — continued

Procedure for electroeluting DNA from agarose gels

16.

Pass electric current through the bag (4 - 5 V/cm for 2 - 3hours). During this time, the DNA is eluted out of the gel
and onto the inner wall of the dialysis tubing. This process
can be monitored with a hand-held, long-wavelength
UV lamp. Expose to UV light briefly.
Reverse the polarity of the current for 1 minute. This will

1.

Electrophorese DNA in an agarose gel.

2.

Stain the gel with GelStar® or SYBR® Green I Nucleic Acid
Stain or ethidium bromide.

3.

Locate the band of interest using a UV light source.
Minimize exposure of DNA to UV light to less than 1
minute.

17.
18.

Recover the bag from the electrophoresis chamber.

4.

Excise the band of interest using a scalpel or razor
blade.

19.

5.

Wet a spatula with 1X TAE Buffer.

6.

Place the agarose slice containing the DNA on a wetted
spatula or a scalpel.

Gently massage the side of the bag with gloved fingers
where the DNA has accumulated. This will remove
the DNA from the wall of the bag. This process can bemonitored with a hand-held UV lamp. Expose to UV lightbriefly.

7.

Photograph the gel for a record of which band waseluted.

20.

Open the dialysis bag.

21.

8.

Seal one end of treated dialysis tubing with a dialysis
clip.

Transfer all of the buffer surrounding the gel slice to aplastic tube.

22.

Wash out the bag with a small amount of 1X TAE Buffer.

9.

Fill the bag to the top with 1X TAE Buffer.

23.

Transfer the solution to the plastic tube.

10.

Transfer the slice of agarose into the bag with the
spatula.

24.

Remove the gel slice from the bag.

25.

optional: Stain the slice with GelStar® or SYBR® Green I
Nucleic Acid Stain or ethidium bromide to ensure all the
DNA has been eluted from the slice.

26.

Purify the DNA from 1X TAE Solution using phenol/
chloroform extractions.*

11.

Allow the slice of agarose to sink to the bottom of the
bag.

12.

Remove the buffer from the dialysis bag, leaving justenough to keep the gel slice in constant contact withthe buffer.

13.

Clip the dialysis bag above the gel slice, avoiding air
bubbles.

14.

Place a shallow layer of 1X TAE Buffer in anelectrophoresis chamber.

15.

Immerse the bag in the electrophoresis chamber.

remove the DNA from the wall of the bag.

Recovery of DNA from
Agarose Gels

Returnto Main*After electroelution, it is recommended that the DNA is further purified
with a phenol/chloroform extraction followed by ethanol precipitation.
Oligosaccharides and other contaminants (found in low-grade agarose) can
copurify with the DNA. Phenol extractions will remove any oligosaccharides,
avoiding their coprecipitation during ethanol precipitations.

References
Bostian, K.A., et al., Anal. Biochem. 95: 174 - 182, 1979.
Dretzen, G., et al., Anal. Biochem. 112: 295 - 298, 1981.
Girvitz, S.D., et al., Anal. Biochem. 106: 492 - 496, 1980.
Henrich, B., et al., J. Biochem. Biophys. Methods 6: 149 - 157, 1982.
Ho, N.W.Y., Electrophoresis 4: 168 - 170, 1983.
Smith, H.O., Meth. Enzymol. 65: 371 - 380, 1980.
Strongin, A.Y., et al., Anal. Biochem. 79: 1 - 10, 1977.
Tabak, H.F. and Flavell, R.A., Nucl. Acids Res. 5: 2321 - 2332,1978.

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Section VI: Recovery of DNA from Agarose Gels
Phenol/Chloroform Extraction of DNA from Agarose Gels
Compatible agaroses–– SeaPlaque® GTG® Agarose (certified and tested forthe recovery of DNA)–– NuSieve® GTG® Agarose (certified and tested for therecovery of DNA)–– SeaPlaque® Agarose

Recovery of DNA from
Agarose Gels

Tips
Recovery failures when extracting DNA from agarose
using phenol/chloroform most often result from either
extracting too large a piece of agarose, or precipitating
agarose along with the DNA at the ethanol precipitation
step. To address these difficulties, we recommend thefollowing:—— No more than 200 mg (200 µl) of agaroseshould be extracted in a single tube;
if your gel slice containing the DNA is larger than this,separate it into smaller pieces, then combine theextracted solutions prior to ethanol precipitation

8.

A dd the appropriate volume of TE Buffer (prewarmed
to 67°C) so that the final concentration of agarose is
0.5%.

9.

Maintain the samples at 67°C until you are ready tophenol extract.

10.

Add an equal volume of buffer-equilibrated phenol.

11.

All subsequent steps can be done at room temperature.

12.

Vortex for 15 seconds.

13.

Centrifuge at maximum speed in a microcentrifuge for3 minutes.

14.

Carefully remove the top aqueous phase. The interfaceof white debris is the agarose, which can contain sometrapped DNA. This can be back-extracted with TE tomaximize yield.

15.

Place aqueous phase in a clean microcentrifuge tube.

16.

Repeat steps 10 - 15.

17.

Add an equal volume of phenol/chloroform to the aqueous

phase.
18.

Vortex for 15 seconds.

—— Ethanol precipitation of agarose can be avoided bychilling the extracted solution on ice for 15 minutes,
then centrifuging the sample(s) in a cold roomfor 15 minutes at maximum speed in a microcentrifuge
prior to adding salts and ethanol. The supernatantis then carefully decanted, and the DNA in the
supernatant is precipitated following standard
protocols

19.

Centrifuge at maximum speed in a microcentrifuge for3 minutes.

20.

Remove the aqueous phase and place in a clean
microcentrifuge tube.

21.

Repeat the extraction with an equal volume ofchloroform.

22.

Remove the aqueous phase and place in a clean—— Not useful for large DNA (>10kb). Vortexing will shear
the DNA

23.

Chill the aqueous phase for 15 minutes on ice.

24.

Centrifuge in a microcentrifuge at high speed for
15 minutes at 4°C.

25.

Carefully decant the supernatant into a clean microcentrifuge tube.

26.

Ethanol precipitate the DNA in the supernatant following
standard protocols.

ProcedureThis protocol has been adapted from Sambrook, et al.
1.

Electrophorese DNA in a low melting temperature
agarose gel prepared in 1X TAE Buffer.

2.

Set a water bath for 67°C.

3.

Prewarm the TE at 67°C.

4.

Excise the gel fragment containing the DNA.

5.

Place the DNA slice in a microcentrifuge tube.

6.

Estimate the volume of the slice. If the slice issignificantly greater than 200 mg, break the agarose
slice into smaller pieces and place each agarose piecein a separate microcentrifuge tube.

7.

Melt the gel slice at 67°C for 10 minutes.

microcentrifuge tube.

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Section VI: Recovery of DNA from Agarose Gels
Phenol/Chloroform Extraction of DNA from Agarose Gels — Continued■■ Materials■■ Reagents–– Water bath set to 67°C–– Microcentrifuge tubes (≥1 ml)–– SeaPlaque® GTG®, NuSieve® GTG® or SeaPlaque®
Agarose–– Ice bucket and ice–– TE Buffer warmed to 67°C–– Vortex mixer–– Buffer-equilibrated penol–– Microcentrifuge–– Chloroform–– Scalpel or razor blade–– Phenol/chloroform (1:1)

Recovery of DNA from
Agarose Gels

Returnto MainCaution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

References
Benson, S.A., BioTechniques 2: 66 - 67, 1984.
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Laboratory Press, 1989.

Section VI: Recovery of DNA from Agarose Gels
"Modified Freeze/Squeeze" Extraction of DNA from Agarose Gels
Introduction

2.

Excise gel slice containing DNA.

This protocol is relatively fast and will work with low andstandard melting temperature agaroses. However, likemany recovery techniques, the DNA yield decreases withincreasing DNA size, but should be approximately 50% for
DNA 5 kb.

3.

Place in a microcentrifuge tube.

4.

Estimate volume based on weight.

5.

Mash the gel slice with the stir rod.

6.

Add an equal volume of buffer-equilibrated phenol.

7.

Vortex for 10 seconds.

8.

Freeze at -70°C for 5 to 15 minutes.

9.

Centrifuge for 15 minutes at room temperature.

Compatible agaroses–– SeaKem® GTG® Agarose (certified and tested for therecovery of DNA)

10.

Remove the supernatant which contains the DNA.

–– SeaPlaque® GTG® Agarose (certified and tested forthe recovery of DNA)

11.

Place the supernatant, in a clean microcentrifuge tube.

–– NuSieve® GTG® Agarose (certified and tested for therecovery of DNA)

12.

Phenol/chloroform extract the supernatant.

13.

Follow with a chloroform extraction.

14.

Ethanol precipitate following standard procedures.

–– SeaPlaque® Agarose–– MetaPhor® Agarose■■ Materials–– NuSieve® 3:1 Agarose–– Microcentrifuge tubes (≥1 ml)–– SeaKem® Gold Agarose–– Ice bucket and ice

Procedure–– Vortex mixer

This protocol has been adapted from Benson. For moredetailed protocols on phenol chloroform extractions, referto the previous section.

–– Scalpel or razor blade

1.

Electrophorese DNA in an agarose gel prepared in 1X TAE
Buffer.

–– Microcentrifuge–– Glass or plastic stir rod■■ Reagents–– TE Buffer warmed to 67°C

References–– Buffer equilibrated phenol

Benson, S.A., BioTechniques 2: 66 - 67, 1984.
Polman, J.K. and Larkin, J.M., Biotechnology Techniques 3: 329 - 332, 1989.
Tautz, D. and Renz, M., Anal. Biochem. 132: 14 - 19, 1983.

–– Chloroform–– Phenol/chloroform (1:1)
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Section VI: Recovery of DNA from Agarose Gels
Ethanol Precipitation of DNA Recovered from Agarose Gels

Introduction
This method is compatible with all the recovery techniques
listed in this chapter.

■■ Materials–– Ice bucket and ice–– Vortex mixer–– Microcentrifuge

Tips

Recovery of DNA from
Agarose Gels—— Prior to adding salts and ethanol, precipitation of
agarose can be avoided by chilling the supernatant on
ice for 15 minutes, then centrifuging the sample(s)in a cold room for 15 minutes at maximum speed ina microcentrifuge. The supernatant is then carefully
decanted, and the DNA in the supernatant is ethanol
precipitated following standard protocols—— Ethanol precipitations should be incubated at room
temperature with ammonium acetate rather thansodium acetate in order to decrease the likelihood ofcoprecipitation of agarose-oligosaccharides with the
DNA or RNA–– Vacuum gel dryer–– Microcentrifuge tubes (≥1 ml)■■ Reagents–– 100% ethanol (ice cold)–– 70% ethanol (room temperature)–– TE Buffer, pH 8.0–– 10 M ammonium acetate

ProcedureThis protocol has been adapted from Sambrook, et al.
1.

Chill the supernatant on ice for 15 minutes.

2.

Centrifuge the sample(s) in a cold room for 15 minutes
at maximum speed in a microcentrifuge.

3.

Carefully decant the supernatant.

4.

Place in a clean microcentrifuge tube.

5.

Measure the volume of the sample.

6.

Add 0.2 volumes of 10 M ammonium acetate to thesample.

7.

Add 2 volumes of 100% ice-cold ethanol.

8.

Briefly vortex.

9.

Store the mixture for 30 minutes to overnight at room
temperature.

10.

Centrifuge for 30 minutes at 12,000 rpm.

11.

Decant the supernatant.

12.

Wash the pellet three times with 70% ethanol.

13.

Allow to air-dry at room temperature on the bench top.

14.

Dry under vacuum for 5 - 10 minutes.

15.

Dissolve the DNA in TE Buffer.

References
Life Technologies, Inc., gibco-BRL Focus 7(4): 1 - 2, 1985.
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory Press, 1989.

References for other recovery methods
References for Electrophoresis onto DEAE-cellulose membrane
Dretzen, et al., Anal. Biochem. 112: 295, 1981.
Girvitz, et al., Anal. Biochem. 106: 492, 1980.
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory Press, 1989.
Winberg, G. and Hammarskjold, M.L., Nucl. Acids Res. 8: 253 - 264, 1980.
Yang, R.C.A., et al., Meth. Enzymol. 68: 176 - 182, 1979.
Zassenhaus, H.P., et al., Anal. Biochem. 25: 125 - 130, 1982.
References for Passage through DEAE-sephacel
Sambrook, J., et al., Molecular Cloning : A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory Press, 1989.
References for Recovery using glass beads
Ausubel, F.M., et al., Current Protocols in Molecular Biology, Red Book, Wiley& Sons, 1995.
Chen, C.W. and Thomas, C.A., Anal. Biochem. 101: 339 - 341, 1980.
Vogelstein, B. and Gillespie, D., Proc. Natl. Acad. Sci. 76: 616 - 619, 1979.
General References
Ausubel, F.M., et al., Current Protocols in Molecular Biology, Red Book, Wiley& Sons, 1995.
Hengen, P.N., Methods and Reagents; Recovering DNA from Agarose Gels.
TIBS 19, 1994.
Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor, Cold Spring Harbor Laboratories, 1982.
Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory Press, 1989.

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