Lonza Rockland, Inc.
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Document # 18121-0807-05
Rockland, ME 04841 USAβ-Agarase

β-Agarase buffer in one hour at 40° C. Similar activities are
obtained for other low-melting-temperature agaroses such as
NuSieve® GTG® Agarose.

Product Description
Lonza β-Agarase, an enzyme preparation derived from
Pseudomonas atlantica, will digest the polysaccharide
backbone of agarose [3, 6-anhydro-α-L-galactopyranosyl(1→ 3)-D-galactose] to alcohol soluble
neoagaro-oligosaccharides.1, 2, 3 DNA or RNA electrophoresedin a low-melting-temperature agarose gel can be quantitatively
recovered after the gel is melted and digested with this enzyme
4, 5
The remaining alcohol soluble neoagaropreparation.
oligosaccharides will not gel or interfere with subsequent DNA
manipulations such as cloning, labeling, restriction digestion, orsequencing. Lonza β-Agarase is free of any detectable
DNase, RNase, or phosphatase activity. β-Agarase isparticularly useful for recovering large DNA (>10 kb) which
could be sheared by other methods of recovery.

Concentration: 1 unit per μl
β-Agarase Buffer
Supplied as a 50X concentrate.
1X concentration is: 40 mM Bis Tris/HCl, 40 mM NaCl, 1 mM
EDTA (pH 6.0).

Agarose Digestion
Before you begin:•Set one water bath or heating block for 70°C. Set anotherwater bath or heating block for 45°C.
•Electrophorese your DNA or RNA sample in a low-meltingtemperature agarose gel (SeaPlaque®, SeaPlaque®
GTG®, orNuSieve® GTG® Agarose) prepared in TAE, TBE, or
MOPS buffer.
•Place no more than 200 mg of an agarose gel slice into a2 ml microcentrifuge tube.

Contaminating Enzyme Activity
Lonza β-Agarase is tested for the presence of contaminating
enzyme activity by incubating 30 units ofβ-Agarase with 1 μg each of the substrates listed below under
the following conditions: 16 hours at 37°C in 50 μl of a bufferconsisting of 33 mM Tris/acetate, 66 mM potassium acetate,
10 mM magnesium acetate, 0.5 mM DTT, and 100 μg/ml BSA,
pH 7.8.
DNase:

RNase:

Phosphatase:

Basic Method
1.

There is no change in the electrophoretic
migration of : Hind III digested λ phage DNA;
super coiled plasmid DNA; a 5‫ ׳‬phosphorylated
40 base oligomer; or a 5‫׳‬-OH 40 base oligomer
indicating the absence of any DNase activity.

2.
3.

There is no change in the electrophoretic
migration of an in vitro generated SP6 RNApolymerase transcript indicating the absenceof any RNase activity.

4.

There is no degradation of p-nitrophenylphosphate indicating the absence of any
phosphatases.

Storage

The enzyme is stable when stored at -20°C.2 β-Agarase issupplied in a storage buffer consisting of 50% glycerol, 50 mM
Tris/HCl, 100 mM NaCl, and 0.1% Triton® X-100 surfactant atpH 7.5

Equilibrate the gel slice with 10 volumes of 1X β-Agarase
buffer for 60 minutes at room temperature, then discard
the buffer. See Application Notes 1.
Melt the gel slice at 70°C (approximately 15 minutes).
Make sure that the gel slice is completely melted as theenzyme will not digest agarose in the gelled state.
Cool the melted gel to 45°C and add 1 unit of β-Agarase
for 200 mg (approximately 200 μl) of 1% agarose gel, mixand incubate at 45°C for 60 minutes.
Add proportionally more or less of the enzyme preparation
with larger or smaller gel slices. Also, add proportionallymore enzyme to agarose gel concentrations higher than1%. For example, 3 units of β-Agarase should be used todigest 200 mg of a 3% agarose gel.
NOTE: The enzyme preparation retains full activity forseveral hours at 45°C. However it will gradually lose
activity upon longer incubations at 45°C. For this reason
we recommend incubation at 40°C for overnightdigestions.

Fast Method: If you wish to save time use the fastequilibration method.

Unit Definition:
One unit of β-agarase will completely digest 200 mg of amolten 1% SeaPlaque® GTG® Agarose Gel prepared in 1X

11.

Melt the gel slice at 70°C, (approximately 15 minutes).
Make sure the gel slice is completely melted as theenzyme will not digest agarose in the gelled state.

2.

Add 2 μl of 50X β-Agarase buffer to every 100 μl of gel
solution.
Mix.
Cool the melted gel to 45°C.
For TAE gels or MOPS gels: add 2 units of β-Agarase for
200 mg (approx. 200 μl) of 1% agarose gel.
For TBE gels: add 7 units of β-Agarase for 200 mg(approx. 200 μl) of 1% agarose gel. Add proportionallymore or less enzyme with larger or smaller gel slices, orwith agarose concentrations higher or lower than 1%. For
example, 21 units of β-Agarase should be used to digest
200 mg of a 3% agarose TBE gel. (approx. 200 μl).

3.
4.
5.

that either carrier tRNA or nuclease free glycogen beadded. In addition, overnight precipitation at room
temperature can be helpful.(7, 8)
3. Please note that polynucleotide kinase is inhibited by(6)When phosphorylating your≥7 mM ammonium ion.
DNA sample with this enzyme, sodium acetate may be abetter choice than ammonium acetate.
4. When recovering RNA from agarose gels, be sure that any
denaturants in the gel (e.g. formaldehyde or glyoxal) arecompletely removed prior to adding the β-Agarase. These
denaturants will interfere with enzyme activity. They can beremoved by equilibrating the gel slice in the β-Agarase
buffer prior to adding the enzyme preparation

DNA Recovery and Purification
For Fragments ≤30 kb••••Make sure the solution has no particles in it; it should lookclear.
Place at -20°C for 30 minutes.
Spin at 4°C for 15 minutes.
Remove the supernatant and place it into a clean
microcentrifuge tube.

References

At this point the sample can be concentrated without ethanol
precipitation by centrifugation in a molecular weight-cutoff-spincolumn.
To Ethanol Precipitate:•Add ammonium acetate to the supernatant to a final
concentration of 2.5 M. The use of ammonium acetate
rather than sodium acetate decreases the likelihood of coprecipitation of agarose-oligosaccharides with the nucleicacid.
•Add 2 to 3 volumes of 100% ethanol.
If the DNA is ≤0.05 μg/ml add 10 μg of carrier RNA andprecipitate at room temperature for 24 hours. SeeApplications Note 2.
If DNA is ≥0.05 μg/ml precipitate at room temperature for
30 minutes.
•
Collect the precipitate by centrifugation. Centrifugation of
the precipitated DNA should be done for a full 30 minutes
at maximum speed (12,000 to 16,000 x g). The
30 minutes centrifugation time is important for smaller
fragments and lower concentrations.

1.

Yaphe, W. (1957). The use of agarase fromPseudomonas atlantica in the identification of agar inmarine algae (Rhodophyceae). Can. J. Microbiol. 3: 987993.

2.

Morrice, L.M., McLean, M.W., Williamson, F.B., and Long,W.F. (1983). β-agarases I and II from Pseudomonas
atlantica. Purification and some properties. Eur. J.
Biochem. 135: 553-558.

3.

Morrice, L.M., McLean, M.W., Long, W.F., and Williamson,F.B. (1983). β-agarases I and II from Pseudomonas
atlantica. Substrate specificities. Eur. J. Biochem. 137:149-154.

4.

Bucan, M., Yang-Feng, T., Colberg-Poley, A.M.,
Wolgemuth, D.J., Guenet, J-L., Francke, U., and Lehrach,
H. (1986). Genetic and cytogenetic localization of thehomeo box containing genes on mouse chromosome 6and human chromosome 7. EMBO J. 5: 2899-2905.

5.

Couto, L.B., Spangles, E.A., and Rubin, E.M. (1989). A
method for the preparative isolation and concentration ofintact yeast artificial chromosomes. Nucleic Acids
Research 17: 8010.

6.

Richardson, C.C. (1981). Bacteriophage T-4rdpolynucleotide kinase. The Enzymes, 3 edition, Vol. 14,
pp. 299-314. Academic Press, New York.

7.

Zeugin, J.A. and Hartley, J.L. (1985). Ethanol
precipitation of DNA. Focus (Life Technologies) 7: 1-2.

8.

Crouse, J. and Amorese, D. (1987). Ethanol precipitation:ammonium acetate as an alternative to sodium acetate.
Focus (Life Technologies). 9: 3-5.

9.

Lamb, B.T., Sisodia, S.S., Lawler, A.M., Slunt, H.H., Kitt,C.A., Kearns, W.G., Pearson, P.L., Price, D.L., andGearhart, J.D. (1993). Introduction and expression of the400 kilobase precursor amyloid protein gene in transgenic
mice. Nature Genetics 5: 22-30.

For Fragments ≥30 kb•

The DNA/β-Agarase solution may be used directly without
precipitation for subsequent enzymatic manipulations.
Alternately the sample can be concentrated without
ethanol precipitation by centrifugation in a molecular(9)weight-cutoff-spin column.

Application Notes
1.

2.

The enzyme preparation has maximum activity between
pH 6 and 7 and is relatively unaffected by salt
concentrations between 0.1 M and 0.25 M.(2) The
equilibration of the gel slice with β-Agarase buffer isnecessary to provide the enzyme with optimal bufferconditions. This equilibration is more important for gels
prepared in TBE than in TAE buffer due to the greaterbuffering capacity of TBE buffer.
For efficient recovery of small nucleic acids (<500 bp)
and/or very dilute samples (>0.5 μg/ml), we recommend

2Ordering Information:
Catalog No.

Description

Size

58001
58005

β-Agarase
β-Agarase

100 units
500 units

For Research Use Only.
Not for use in Diagnostic Procedures.

Related Products
SeaPlaque® Agarose
SeaPlaque® GTG® Agarose
NuSieve® GTG® Agarose
For more information contact Technical Service at (800)521-0390 or visit our website at www.Lonza.com.

Triton is a trademark of Rohm & Haas Company. All other trademarks
herein are marks of the Lonza Group or its affiliates. Manufactured forLonza© 2007 Lonza Rockland, Inc.
All rights reserved.

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