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β-Agarase I |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- DNase and RNase Free
- Supplied with 10X Reaction Buffer
Description:
β-Agarase cleaves the agarose subunit, unsubstituted neoagarobiose [3,6-anhydro-a-L-galactopyranosyl-1-3-d-galactose] to neoagaro-oligosaccharides (1).
Source:
Isolated from a strain of E. coli that carries a plasmid which encodes the β-Agarase I gene.
Applications:- β-Agarase I digests agarose, releasing trapped DNA and producing carbohydrate molecules which can no longer gel. β-Agarase I can be used to purify both large (> 50 kb) and small (< 50 kb) fragments of DNA from gels. The remaining carbohydrate molecules and β-Agarase I will not, in general, interfere with subsequent DNA manipulations such as restriction endonuclease digestion, ligation, and transformation.
Reagents Supplied:
β-Agarase I Reaction Buffer (10X)
Reaction & Storage Conditions
Reaction Conditions:
1X β-Agarase I Reaction Buffer
Incubate at
42°C.
1X β-Agarase I Reaction Buffer:
10 mM Bis-Tris-HCl
1 mM EDTA
pH 6.5 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 200 μl of molten low melting point or NuSieve agarose to nonprecipitable neoagaro-oligosaccharides in 1 hour at 42°C.
Concentration:
1,000 units/ml
Storage Conditions:
50 mM Bis-Tris-HCl
1 mM EDTA
50% Glycerol
pH 6.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- β-Agarase I retains activity for several hours at 40-45°C and is stabilized by the presence of agarose in the reaction.
Usage notes:- Only low melting point agarose is suitable for β-Agarase I digestion as the solution must be liquid at the incubation temperature of 42°C. If the temperature falls below 42°C during the reaction time, even low melting point agarose will begin to congeal and be undigestable.
- β-Agarase I is quickly inactivated at temperatures above 45°C. Therefore, when working with large volumes, be sure to leave ample time for the molten agar to equilibrate to 42°C.
- β-Agarase I works best on gels made with Tris-acetate buffer (TAE). For gels made with Tris-borate buffer (TBE), doubling the required amount of β-Agarase I is recommended.
- β-Agarase I works most efficiently on solutions containing 1% agarose or lower. For maximum digestion of higher percentage gels, melt the gel slice at 65°C and adjust the volume with 1X β-Agarase I Buffer so that the final concentration of agarose is 1%.
- β-Agarase I exhibits optimal activity at pH 6.5. Greater than 75% of the optimal activity is maintained between pH 5.0-8.5.
- Incubation at 95°C for 2 minutes or incubation at 65°C for 15 minutes inactivates 50 units of β-Agarase I. β-Agarase I retains activity for several hours at 40°-45°C and is stabilized by the presence of agarose in the reaction.
FAQs
- Can ligation and other DNA manipulations be carried out on β-Agarase I treated gel slices containing DNA?
- What is the molecular weight of β-Agarase I?
- What type of agarose will β-Agarase I digest?
- Will β-Agarase I work in other buffers?
- Why does a white precipitate form after the reaction using β-Agarase I?
- Can a high percentage low melt gel be digested with β-Agarase I?
- What is a common cause of β-Agarase I reaction failure?
- Does the gel running buffer have an effect on the β-Agarase I reaction?
- How can β-Agarase I be heat inactivated?
- How stable is β-Agarase I in reaction?
- What is the optimal pH for β-Agarase I digestion?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of contaminating endonucleases, exonucleases and ribonucleases.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA (HindIII digest)
and 16 unit of β-Agarase I incubated for 16 hours at 42ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 10 units of β-Agarase I with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 42ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 8 units of β-Agarase I with 1 μg of
ΦX174 RF I DNA for 4 hours at 42ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
RNase Assay:
Incubation of a 50 μl reaction containing 4 units of β-Agarase I with 2
μg of RNA Ladder for 1 hour at 42ºC
resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
Recovery of DNA from LMP agarose after β-Agarase I Treatment: 1 µg of BstNI-digested pBR322 DNA in 200 µl of 1% LMP agarose was melted at 65°C, mixed with 22 µl of 10X β-Agarase I Reaction Buffer and cooled to 42°C. 1 unit of β-Agarase I was added, mixed, and incubated at 42°C for 1 hr. DNA was precipitated by adding NaOAc to 250 mM, isopropanol to 75%, and chilling at -70°C for 1 hour. DNA was recovered by centrifugation, resuspended in TE, and electrophoresed on a 1% gel. Lane 1: BstNI-digested pBR322 DNA (control). Lane 2: BstNI-digested pBR322 DNA purified using β-Agarase I.
Alcohol Precipitable Agarose after β-Agarase I Treatment: Time course of alcohol precipitable carbohydrate after β-Agarase I treatment of low melting point (LMP) agarose: Samples containing 200 µl of 1% LMP agarose in 10 mM Bis Tris-HCl, 1 mM Na2EDTA were melted at 65°C, cooled to 42°C, and incubated with 1 unit or 2 units of β-Agarase I. Undigested agarose was precipitated by adding NaOAc to 250 mM, isopropanol to 75% and chilling at -70°C. Precipitated agarose was hydrolyzed by boiling in 0.1N HCl, and carbohydrates were measured by the method of Dygert, et al (3). Remaining carbohydrate is expressed as a percentage of total precipitable carbohydrate in 200 µl of undigested LMP agarose.
References
- Yaphe, W. (1957) Can. J. Microbiol., 3, 987-993.
- Davis, T. and Guan, C., unpublished observations.
Reagents Sold Separately
β-Agarase I Reaction Buffer
Companion Products
β-Agarase I Reaction Buffer
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