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DNA Modifying Enzymes and Cloning >
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Micrococcal Nuclease |
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Prices are in US dollars and valid only for US orders.
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- Can be used for Chromatin Studies
- Inactive until Ca2+ is added
Description:
Micrococcal nuclease is derived from Staphylococcus aureus and is a relatively non-specific endo-exonuclease. It is purified from a recombinant E. coli strain that digests double-stranded, single-stranded, circular and linear nucleic acids. The enzyme is active in the pH range of 7.0 - 10.0, with optimal activity at pH 9.2 for both RNA and DNA substrates. Cleavage preferences have been observed at sites rich in adenylate, deoxyadenylate or thymidylate (1). Both DNA and RNA are degraded to 3´ phosphomononucleotides and dinucleotides.
Source:
An E. coli strain containing a genetic fusion of the micrococcal nuclease gene (Gene ID: 3238436) and the gene coding for maltose binding protein, or MBP. The micrococcal nuclease is cleaved from the fusion protein and purified away from MBP.
Applications:- Degrade nucleic acids present in protein preparations
- In vitro translation (2)
- Reduce the viscosity of cell lysates during non-mechanic cell lysis preparation
- Chromatin structure analysis (3)
- Rapid RNA sequencing
Reagents Supplied:
Micrococcal Nuclease Reaction Buffer (10X)
BSA (100X)
Enzyme Properties
Nuclease Properties Comparison
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X Micrococcal Nuclease Reaction Buffer
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X Micrococcal Nuclease Reaction Buffer:
50 mM Tris-HCl
5 mM CaCl2
pH 7.9 @ 25°C
Unit Definition:
(Kunitz Unit) One unit is defined as the amount of enzyme required to release acid soluble oligonucleotides that produce an absorbance increase of O.D. 1.0 at 260 nm in 30 minutes at 37°C.
(Agarose Gel Unit) One gel unit is defined as the amount of enzyme required to digest 1 µg of lambda genomic DNA in 15 minutes at 37°C, to the extent that the accumulation of low molecular DNA fragments (100-400 base pairs) disappears on a 1.2% agarose gel.
Note: 10,000 Gel Units is approximately equal to 1,000 Kunitz Units.
Unit Assay Conditions:
(Kunitz Unit) 1X Micrococcal Nuclease Buffer, 0.1mg/ml BSA and 500 µg sonicated Salmon testis genomic DNA in a total volume of 500 µl.
(Agarose Gel Unit) 1X Micrococcal Nuclease Buffer, 0.1mg/ml BSA and 1 µg of Lambda genomic DNA in a total volume of 50 µl.
Concentration:
2,000,000 gel units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM NaCl
1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- This enzyme does not work in NEBuffer 1, 2, 3 or 4 due to the lack of Ca2+. Additional Ca2+ in NEBuffer only shows 10% activity. 1-5 mM Ca2+ is required for activity.
The enzyme is active in the pH range 7-10 as long as salt concentration is less than 100 mM.
Enzyme can be inactivated by addition of excess EGTA.
FAQs
- Will Micrococcal Nuclease cleave DNA as well as RNA and what type of end is left after 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:
Free of detectable protease activity.
Figure 1: Digestion of 1 µg of Lambda genomic DNA with Micrococcal Nuclease in a 3-fold dilution series. The amount of enzyme used in Lane 2 is defined as 1 gel unit. Lane M is the PCR Marker (NEB #N3234).
References
- Cuatrecasas, S.F., and Anfinsen, C.B. (1967) J. Biol. Chem., 244, 1541-1547.
- Craig, D. et al. (1992) Nucl. Acids Res., 20, 4957-4987.
- O'Neill, L.P. and Turner B.M. (2003) Methods, 31, 76-82.
Reagents Sold Separately
BSA
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