Shipping and Storage

Ice pack transportation; Stored at -20℃, with a shelf life of 2 years, to avoid repeated freezing and thawing.

Component

Description

Exonuclease I is derived from the recombinant expression of the Exo I gene in E. coli strains. This enzyme has the activity of hydrolyzing single stranded DNA from the 3-5 “direction, gradually releasing deoxyribonucleic acid 5” monophosphate and leaving intact 5 “terminal dinucleotides. This product is mainly used for the degradation and digestion of primers after PCR amplification, and is inactive for double stranded DNA and 3OH terminal DNA chains blocked by phosphoryl or acetyl groups.

Application

Commonly used to remove single stranded primers in PCR reactions before Sanger sequencing or SNP analysis; Remove single stranded primers from nested PCR; Purification of PCR products by enzymatic method; Remove single stranded DNA containing 3 ‘hydroxyl terminus from nucleic acid mixtures.

Specification

1. Source: Escherichia coli strain carrying the exonuclease I gene cloned from E. coli NM554.
2. Enzyme storage solution: 10mM Tris HCl, 100mM NaCl, 5mM β – ME, 0.5mM EDTA, 100µg/ml BSA, 50% Glycerol pH 7.5 @ 25℃.
3. Unit definition: 1 unit refers to the amount of enzyme required to catalyze the release of 10nmol of acid soluble nucleosides within 30 minutes at 37℃.
4. Inactivation or inhibition: react at 80℃ for 20 minutes.

Product Number: S120308

Shipping and Storage

Wet ice transportation; -Stored at 20℃, with a validity period of 12 months.

Component

Description

Lambda Exonuclease, also known as lambda Exonuclease or lambda exonuclease, is a 5 ‘-3’ deoxyribonuclease derived from the exonuclease gene of lambda bacteriophages and recombinant expressed in Escherichia coli. It can specifically hydrolyze the 5 ‘end phosphorylated strand in double stranded DNA along the 5’ -3 ‘direction, but has low enzymatic activity for single stranded DNA and non phosphorylated dsDNA at the 5’ end, and cannot be digested from DNA cuts or gaps.

Application

Production of single stranded PCR products, analysis of DNA single stranded conformational polymorphism, rolling ring amplification, PCR product cloning, etc.

Features

1. Source: Derived from λ bacteriophage, recombinant expressed by Escherichia coli.
2. Enzyme activity definition: The amount of enzyme required to hydrolyze 10nmol of acid soluble deoxyribonucleotide from a double stranded DNA substrate in a 50μL reaction system at 37℃ within 1 minute is defined as one enzyme activity unit.
3. Purity and concentration: SDS-PAGE detection shows a purity of ≥ 95%; Endogenous nucleic acid residue<1 pg/μL (qPCR detection); Enzyme activity is 5U/μL.
4. Inactivation or inhibition: Incubate at 75℃ for 10 minutes to inactivate.
5. Enzyme storage buffer: 25mM Tris HCl, 50mM NaCl, 1 mM DTT, 0.1mM EDTA, 50% Glycerol, pH 8.
6. 10×Reaction Buffer: 670mM Glycine-KOH, 25mM MgCl₂, 500μg/mL BSA, pH 9.4.

Product Number: EN03

Description

Exonuclease III is an enzyme that acts on double-stranded DNA, progressively removing single nucleotides from the 3′-OH terminus. The optimal substrates for this enzyme are blunt-ended or 5′ overhanging DNA, but it can also act on double-stranded DNA nick sites to produce single-strand nicks. Since it is inactive on single-stranded DNA, it has difficulty cleaving 3′ overhanging ends. The 3′ to 5′ exonuclease activity of Exonuclease III varies with the length of the 3′ overhang; ends with four or more nucleotides are difficult to cleave. This characteristic can be used to produce single-stranded DNA in a specific direction. For example, linearized DNA can be designed with one end being an uncleavable terminus (3′ overhang) and the other end being a cleavable terminus (blunt end or 5′ overhang). In this case, Exonuclease III will digest only one strand.

The activity of Exonuclease III is partly dependent on the DNA double helix structure and varies with the sequence (C > A = T > G). Additionally, this enzyme also exhibits RNase H, 3′-phosphatase, and apurinic/apyrimidinic (AP) site-specific endonuclease activities.

Components

Storage

Store at -30 to -15°C. Shipping conditions: ≤0°C.

Unit Definition

One unit (U) of enzyme activity is defined as the amount of enzyme required to produce 1 nmol of acid-soluble material from double-stranded DNA at 37°C for 30 minutes.

Applications

1. Unidirectional nested deletion
2. Site-directed mutagenesis
3. Preparation of single-strand-specific probes
4. Preparation of single-strand substrates for dideoxy sequencing

Protocol

1 Prepare the reaction mixture as suggested below (on ice):

2 Incubate at 37°C for 30 minutes.

3 Terminate the reaction by adding EDTA to a final concentration of 11 mM, or inactivate Exonuclease III by incubating at 70°C for 30 minutes.

4 Analyze the double-stranded DNA digestion results by agarose gel electrophoresis.

Notes

Exonuclease III cannot cleave thiophosphate bonds. Therefore, a DNA molecule can be protected at one end by introducing an α-thiophosphate nucleotide, allowing for unidirectional digestion. This characteristic can be used for unidirectional digestion of linear DNA molecules with one end being a resistant terminus (3′ overhang) and the other end being a sensitive terminus (blunt end or 5′ overhang).