Klenow Fragment (3′→5′ exo-)

• 产品编号：M0212S      品牌：New England Biolabs       原厂货号：M0212S
• 产品分类：克隆 > 聚合酶 > DNA聚合酶
• 应用分类：

描述：

• Isolated from a recombinant source

• Generates probes using random primers

• Dideoxy sequencing

• Supplied with 10X Reaction Buffer

• Moderate strand displacement activity

Description:
Klenow Fragment (3´→ 5´ exo- ) is an N-terminal truncation of DNA Polymerase I which retains polymerase activity, but has lost the 5´→ 3´ exonuclease activity and has mutations (D355A, E357A) which abolish the 3´→ 5´ exonuclease activity (1).

Source:
An E. coli strain containing a plasmid with a fragment of the E.coli polA (D355A, E357A) gene starting at codon 324.

Applications:

• Random priming labeling

• DNA sequencing by the Sanger dideoxy method (2)

• Second strand cDNA synthesis

• Second strand synthesis in mutagenesis protocols (3).

Reagents Supplied:
NEBuffer 2 (10X)
 

3´ to 5´ Exonuclease: No
5´ to 3´ Exonuclease: No
Strand Displacement: Yes
Error Rate: 100 x10^-6 bases

Heat Inactivation:
75°C for 20 minutes

Molecular Weight:
Theoretical: 68,000 daltons

Reaction Conditions:
1X NEBuffer 2
Supplemented with 33 μM dNTPs (not included)
Incubate at 37°C.

1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl₂
1 mM Dithiothreitol
pH 7.9 @ 25°C

Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.

Unit Assay Conditions: 1X NEBuffer 2, 33 µM dNTPs including [³H]-dTTP and 70 µg/ml denatured herring sperm DNA.

Concentration:
5,000 units/ml and 50,000 units/ml

Storage Conditions:
25 mM Tris-HCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C
 

原厂资料：

• Isolated from a recombinant source

• Generates probes using random primers

• Dideoxy sequencing

• Supplied with 10X Reaction Buffer

• Moderate strand displacement activity

Description:
Klenow Fragment (3´→ 5´ exo- ) is an N-terminal truncation of DNA Polymerase I which retains polymerase activity, but has lost the 5´→ 3´ exonuclease activity and has mutations (D355A, E357A) which abolish the 3´→ 5´ exonuclease activity (1).

Source:
An E. coli strain containing a plasmid with a fragment of the E.coli polA (D355A, E357A) gene starting at codon 324.

Applications:

• Random priming labeling

• DNA sequencing by the Sanger dideoxy method (2)

• Second strand cDNA synthesis

• Second strand synthesis in mutagenesis protocols (3).

Reagents Supplied:
NEBuffer 2 (10X)
 

3´ to 5´ Exonuclease: No
5´ to 3´ Exonuclease: No
Strand Displacement: Yes
Error Rate: 100 x10^-6 bases

Heat Inactivation:
75°C for 20 minutes

Molecular Weight:
Theoretical: 68,000 daltons

Reaction Conditions:
1X NEBuffer 2
Supplemented with 33 μM dNTPs (not included)
Incubate at 37°C.

1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl₂
1 mM Dithiothreitol
pH 7.9 @ 25°C

Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.

Unit Assay Conditions: 1X NEBuffer 2, 33 µM dNTPs including [³H]-dTTP and 70 µg/ml denatured herring sperm DNA.

Concentration:
5,000 units/ml and 50,000 units/ml

Storage Conditions:
25 mM Tris-HCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C
 

注意事项：

Usage notes:

Klenow Fragment (3´→ 5´ exo- ) is not suitable for generating blunt ends because it lacks the 3´→ 5´ exonuclease necessary to remove non-templated 3´ additions.

Klenow Fragment (3´→ 5´ exo–) is also active in all four NEBuffers when supplemented with dNTPs.

Application notes:

When Klenow Fragment (3´→ 5´ exo- ) is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 µl reaction volume is recommended.