Nippon Gene
Nucleic acid extraction from agarose gel

Thermostable β- Agarase
Code No. 317-07123（30 units）, 311-07121（300 units）
Manual (ver.4) 20120105KH

I

Product description

IV Protocol

Agarase is an enzyme which degrades melted (solated) agarose toneoagaro-oligosaccharides, while the agarose solution after thedegradation does not form a gel again. This property allows Agarase
to be used for extracting nucleic acid from agarose gel.
The advantages of nucleic acid extraction by agarase include thefollowing: (1) manipulations are simple, (2) hazardous reagents are
not used, (3) relatively large DNA fragments can be recovered withoutdamage.
This product is an agarase derived from a heat-resistantmicroorganism and has excellent heat resistance compared toconventional agarase.
< Properties >Manipulations are simple and quick.
The optimum reaction temperature is high, and this product canbe used for standard agarose.
The degraded gel solution can be used as-is for reactions such as
cloning and restriction enzyme reactions.
Large DNA fragments can be recovered with minimal shearing.

Basic protocol(1) Put 200 mg (～200 µl) of excised agarose gel block in a tube.
↓(2) Heat for 5-10 min to completely melt agarose gel. *1↓*3(3) After confirming that agarose is melted, transfer to a 50-65°C
constant temperature bath (cool by standing for 30-60 sec). *2↓(4) Add 6 units (6 µl）of Thermostable β-Agarase and mix. *4↓(5) Incubate at 50-65°C for 5-10 min to degrade agarose. *5↓(6) Allow to stand on ice to confirm that the solution does not
re-coagulate. *6(Perform alcohol precipitation/purification as necessary) *7

This enzyme is derived from a heat-resistant*2 Thermostable β-Agarase demonstrates the maximum activity at
50-60°C (left figure). Also, Thermostable β-Agarase keeps 10%
or higher activity after heating at 70°C for 30 min (right figure:arrow).

microorganism collected from the deep sea by
the manned research submarine “Shinkai 6500”
as part of research by the Extremobiosphere*1 The melting point of the gel depends on the kind of agarose andbuffer.

Research Program Institute of Biogeosciences

Heat resistance

Optimum temperature

Chapter 5, Search for useful enzymes from deep

Provided by the Japan Agency forsea microorganisms p.41-52

Marine-Earth Science and Technology(authors:YujiHatada, Yukari Ohta, Yuko Hidaka, Yuichi Nogi)

Relative activity (%)

Technology. For more details, please refer toResidual activity (%)

Japan Agency for Marine-Earth Science andin “Current Technologies of Development andApplication of Enzymes”, CMC Publications.
Reaction temperature (°C)

Data provided by the Japan Agency for Marine-Earth Science and Technology

II Product content
Composition： Thermostable β-Agarase （30 units / 300 units）×1
Activity： 1 unit/ µl
Storage Conditions： 50 mmol/l NaCl, 20 mmol/l Tris-HCl (pH 7.5)
Definition of Units：
One unit is defined as an enzyme activity that produces reducing
sugar equivalent amount of 1 µmol D-galactose from agarose gel per
1 min at 60°C.

Storage temperature： refrigerated （2-10°C）

100

R es idual activity （%）

R e s id u a l a c tiv ity （ % ）

*6

1 00

80
60
40

-20℃
4℃
room temperature

2015

Storage time (Month)20258060
4020010*4 The amount of enzyme to be used can be reduced (see thesection "VI Q&A").

Stability with regard to freezing and thawingStorage stability
1205*3 In the case of low melting point agarose (gel melting point of 65°Cor lower, TAE being used) these steps (2)/(3) can be omitted.

*5 The reaction time varies depending on the amount of agarose gel
block and the container size and the amount of Thermostable
β-Agarase being added.

III Storage

0Heat treatment temperature (°C)

30

If no coagulation is observed in ice cold water, it can bedetermined that agarose is completely degraded.
When
recovered DNA solution is to be used for electrophoresis, theenzyme should be inactivated by heat treatment (90°C, 5 min) or
by a phenol/chloroform treatment.

0020406080

Frequency of freezing and thawing (Time)

10 0120*7 If undegraded gel remains, remove the gel by centrifuge, or restart
the reaction from the start of the protocol. If necessary, perform
alcohol precipitation/purification.

NIPPON GENE CO., LTD. 2-7-18 Toiya-machi, Toyama, 930-0834 TEL: 076-451-6548
URL https://www.nippongene.com/

V Troubleshooting

amount of TE buffer.

Problems and countermeasures

Q5. Can high molecular weight DNA fragments be recovered?
A5. We have confirmed that large DNA fragments can be recovered
with much less physical shearing (confirmed by recovering DNA
fragments of tens of bp to 166 kbp) with Thermostable β-Agarase.
However, the bigger the size of the DNA to be manipulated, the more itis difficult to separate by agarose gel electrophoresis, and the more
prone it will be to shearing by manipulations such as pipetting.

Undegraded gel remaining
Insufficient reaction of Thermostable β-Agarase may be the cause.
1. Add Thermostable β-Agarase after confirming that the gel iscompletely melted in step (2) (gel-dissolving reaction) of the basic
protocol by pipetting, etc.
2. Increase the amount of Thermostable β-Agarase to be added.
3. Extend the reaction time of the gel degradation after adding
Thermostable β-Agarase.
Low purity of recovered DNA
High molecular weight degradation products by agarose may remain.
1. Completely degrade the gel.
2. Precipitate the undegraded substance by centrifuge and use thesupernatant as the DNA solution.
3. Perform alcohol precipitation/purification.
Perform phenol/
chloroform treatment for further purification.
Thinning of the gel around the well in the electrophoresis of therecovered DNA
Thermostable β-Agarase may be persisting.
1. Inactivate by performing a heat treatment at 90°C or higher for 5
min or longer.
2. Perform phenol/chloroform treatment.
VIQ&A

Q1. Can the amount of the enzyme to be employed be reduced?
A1. The amount of the enzyme recommended by the standard
protocol of Thermostable β-Agarase is 6 µl (6 units) per 200 mg gel
block, but the amount of the enzyme to be employed for one reaction
can be reduced depending on the gel concentration and the reaction
condition.
Reaction time

Agaroseconcentration

Amount of enzyme
added

1% Agarose S

2 µl (2 units)

5 min treatment

1.5% Agarose S

3 µl (3 units)(agarose gel：200 mg)

2% Agarose S

5 µl (5 units)

10 min treatment(agarose gel：200 mg)

1.5% Agarose XP

3 µl (3 units)

1.5% Agarose S

1.5 µl (1.5 units)

2% Agarose S

3 µl (3 units)

3% Agarose 21

5.5 µl (5.5 units)

Q2. What is the rate of DNA recovery?
A2. In principle, all DNA in the excised gel is recovered.
Q3. What is the final volume of the reaction solution?
A3. When 200 mg of agarose gel is used, the final volume of thesolution is about 200 µl.
Q4. How can the reaction solution be concentrated?
A4. Perform alcohol precipitation.
Example: Add 0.1 volume of 3 mol/l sodium acetate to the obtained
DNA solution (degraded gel solution) (add the coprecipitating agent,Ethachinmate as necessary, and mix).
Add 0.8-1 volume ofisopropanol (or 2.5 volumes of ethanol), mix, leave standing at room
temperature for 2-10 min, centrifuge (12 K X g, 10 min) and remove
the supernatant. Add about 1 ml of 70% ethanol to the tube
containing the precipitates, centrifuge (12 K X g, 5 min) and remove
the supernatant.
Leave the precipitates standing at room
temperature for 15 min to dry and then dissolve into an appropriate
NIPPON GENE CO., LTD.

Q6. Can the reaction solution be used for PCR reactions?
A6. We have confirmed that the solution can be used for PCR
reactions by adding not more than 1/8 volume of the agarose solution
degraded by Thermostable β-Agarase.
Example: When the volume of the PCR reaction solution is 20 µl, add
2.5 µl or less of the degraded agarose solution.
Q7. Can the reaction solution be used for cloning?
A7. We have confirmed that DNA solution recovered by Thermostable
β-Agarase can be used as-is for ligation and transformation.
(Confirmed by using the Ligation - Convenience Kit and ECOS™
Competent E. coli DH5α.）
Q8. Can the reaction solution be used for transcription reactions?
A8. We have confirmed that DNA solution recovered with
Thermostable β-Agarase can be used as-is for transcription reaction.
(Confirmed by using the CUGA®7 in vitro Transcription Kit)
Q9. Can the reaction solution be used for sequencing?
A9. We have confirmed that DNA solution recovered by Thermostable
β-Agarase can be used as-is for sequencing. （Confirmed by using a377 Sequencer, ABI Inc,）
However, if high-concentration/high molecular weight degradedproducts of agarose is mixed in, there may be problems depending on
the model of the analytical instrument. High molecular weightdegraded products of agarose gel can be removed with aphenol/chloroform treatment.
Q10. Can this product be used for extracting RNA from denaturing
agarose gel?
A10. It can be used, but since formaldehyde included in denaturing
agarose gel block inhibits the enzyme activity, the amount of theenzyme to be added needs to be increased.
Example: After melting 100 mg of 1.5% Agarose S denaturing gel
block at 90°C for 5 min, agarose is degraded by adding 8 µl (8 units) of
the enzyme and reacting at 60°C for 10 min.

For more details on experimental examples and thelike, see the website of Nippon Gene.
Reagents for genetic engineering research,
NIPPON GENE CO., LTD.
https://www.nippongene.com/index.html(Caution)• It is strictly forbidden to reprint, copy, process or distribute without permission
the images, descriptions, data etc., provided by the Japan Agency for
Marine-Earth Science and Technology in this manual.
• This product is sold as a reagent (for investigational research) and must not
be used as a pharmaceutical.

Thermostable β-Agarase Manual（ver.4） 20120105KH