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Nuclease BAL-31 |
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Prices are in US dollars and valid only for US orders.
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- Progressive shortening of duplex DNA
- Inactivation by treatment with EGTA
- Supplied with 2X Reaction Buffer
Description:
Nuclease BAL-31 exonuclease degrades both 3' and 5' termini of duplex DNA without generating internal scissions. The enzyme is also a highly specific single-stranded endonuclease which cleaves at nicks, gaps and single-stranded regions of duplex DNA and RNA (1,2).
Source:
Purified from the culture medium of Alteromonas espejiana BAL-31. Contains a mixture of "fast" and "slow" species of the enzyme (3).
Applications:- Progressive shortening of double-stranded DNA fragments at both termini (4)
- Restriction site mapping (2)
Reagents Supplied:
Nuclease BAL-31 Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
65°C for 10 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X Nuclease BAL-31 Reaction Buffer
Incubate at
30°C.
1X Nuclease BAL-31 Reaction Buffer:
20 mM Tris-HCl
600 mM NaCl
12 mM CaCl2
12 mM MgCl2
1 mM EDTA
pH 8.0 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to remove 200 base pairs from each end of linearized double-stranded ΦX174 DNA (40 µg/ml) in a total reaction volume of 50 μl in 10 minutes at 30°C in 1X Nuclease BAL-31 Reaction Buffer.
Concentration:
1,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM NaCl
1.5 mM CaCl2
1.5 mM MgCl2
0.25 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Duplex products of the exonuclease are a mixture of blunt and staggered ends. This mixture can be cloned directly, although maximal ligation efficiency requires repairing the staggered ends with a suitable DNA polymerase.
- If necessary, the enzyme may be diluted in reaction buffer prior to use.
- Activity is linear with enzyme concentration.
- Heat inactivated by incubation at 65°C for 10 minutes in the presence of 20 mM EGTA, a specific chelator of the essential cofactor Ca2+. This treatment does not affect the Mg2+ concentration.
FAQs
- Can Nuclease BAL-31 be used to remove 10 base pairs from the end of a DNA fragment?
- Why does all of the DNA get degraded when I use Nuclease BAL-31?
- Can Nuclease BAL-31 be heat inactivated?
- Is Nuclease BAL-31 active in other NEBuffers?
- Can Nuclease BAL-31 treated DNA be cloned?
- What is a good control for the BAL-31 nuclease?
- Will Nuclease BAL-31 degrade RNA?
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 detectable double-stranded endonuclease activity.
Double-stranded Endonuclease Activity:
Incubation of 60 units of Nuclease BAL-31 with 65 µg l DNA for 10 minutes at 30°C in 100 µl reaction buffer (rendering 50% of the DNA acid-soluble) resulted in no detectable endonuclease activity. This is judged by the integrity of the internal λ DNA fragments produced by subsequent digestion with Hind III endonuclease.
Ligation of Nuclease BAL-31 treated fragments
A) Gel electrophoresis of Lambda DNA-HaeIII digest
B) Lambda DNA-HaeIII digest after 2 minute incubation with one unit of Nuclease BAL-31
C) As in (B) followed by incubation with T4 DNA Ligase
References
- Gray, H.B. et al. (1975) Nucl. Acids Res., 2, 1459-1492.
- Legerski, R.J. et al. (1978) Nucl. Acids Res., 5, 1445-1463.
- Wei, C.-F. et al. (1983) J. Biol. Chem., 258, 13506-13512.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd Ed.), 5.73-5.75.
Reagents Sold Separately
Nuclease BAL-31 Reaction Buffer
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