Exonuclease III (E. coli)

产品编号：M0206L 品牌：New England Biolabs 原厂货号：M0206L
产品分类：克隆 > DNA/RNA修饰酶 > 核酸酶
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包装：

运保温度:

-20°C

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描述：

• Isolated from a recombinant source

• 3'→5' exonuclease

• Produces unidirectional nested deletions

• Site-directed mutagenesis

• Supplied with 10X Reaction Buffer

Description:
Catalyzes the stepwise removal of mononucleotides from 3´-hydroxyl termini of duplex DNA (1). A limited number of nucleotides are removed during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules (2).

The preferred substrates are blunt or recessed 3´-termini, although the enzyme also acts at nicks in duplex DNA to produce single-strand gaps. The enzyme is not active on single-stranded DNA, and thus 3´-protruding termini are resistant to cleavage. The degree of resistance depends on the length of the extension, with extensions 4 bases or longer being essentially resistant to cleavage. This property can be exploited to produce unidirectional deletions from a linear molecule with one resistant (3´-overhang) and one susceptible (blunt or 5´-overhang) terminus (3).

Exonuclease III activity depends partially on helical structure (4) and displays sequence dependence (C>A=T>G; ref. 5). Temperature, salt concentration and the ratio of enzyme to DNA greatly affect enzyme activity, requiring reaction conditions to be tailored to specific applications.

Exonuclease III has also been reported to have RNase H, 3´-phosphatase and AP-endonuclease activities (1).

Source:
Purified from E.coli K-12, BE257/pSGR3 strain (kindly supplied by B. Weiss)

Applications:

Unidirectional nested deletions (3)

Site-directed mutagenesis (6)

Preparation of strand-specific probes (2)

Preparation of single-stranded substrates for dideoxy sequencing (7)
Reagents Supplied:
NEBuffer 1 (10X)

Heat Inactivation:
70°C for 20 minutes

Specific Activity:
150,000 units/mg

Reaction Conditions:
1X NEBuffer 1
Incubate at 37°C.

1X NEBuffer 1:
10 mM Bis-Tris-Propane-HCl
10 mM MgCl₂
1 mM Dithiothreitol
pH 7.0 @ 25°C

Unit Definition:
One unit is defined as the amount of enzyme required to produce 1 nmol of acid-soluble total nucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X NEBuffer 1 with 0.15 mM sonicated duplex [³H]-DNA.

Concentration:
100,000 units/ml

Storage Conditions:
5 mM KPO₄
200 mM KCl
5 mM 2-Mercaptoethanol
0.05 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 6.5 @ 25°C