Qiagen, X8030L, Lambda Exonuclease (10,000 U)

10,000 U (evaluation pack) Lambda Exonuclease (2.0 ml at 5,000 U/ml) and 10X Lambda Exo Reaction Buffer (1 x 1.5 ml).

Features

- Highly processive 5ʹ→ 3ʹ exonuclease, active on blunt and 5′ recessed ends

Product Details

Lambda Exonuclease is a highly processive 5ʹ→3ʹ double-stranded exonuclease that degrades one strand of the duplex. Lambda exonuclease can initiate a blunt DNA or DNA containing 3ʹ single-stranded overhangs. Lambda Exonuclease has greatly reduced activity on non-phosphorylated DNA, single-stranded DNA and DNA having protruding 5ʹ single-stranded termini. Lambda exonuclease will not initiate at a nick or gap (1,2).

Supplied in: Lambda Exonuclease is supplied in 25 mM Tris·Cl, 50 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA and 50% glycerol: pH 7.5 at 25°C.

Supplied with: 10X Lambda Exo Reaction Buffer (cat. no B8030) contains 670 mM glycine and 25 mM MgCl 2 , pH 9.4 at 25°C.

Performance

Test: Amount tested
Purity: n/a
Specific activity: n/a
Double-stranded endonuclease: 1500 U
E. coli DNA contamination: 1500 U

- Storage temperature: –25°C to –15°C
- Molecular weight: 25,909 Dalton

Principle

The protein is produced by a strain of E. coli that overexpresses the exonuclease gene from bacteriophage Lambda.

One unit is defined as the amount of enzyme required to produce 10 nmol of acid-soluble deoxyribonucleotide from double-stranded substrate in 30 minutes at 37°C.

Procedure

Components: Final Concentration
Nuclease-free water: N/A
10X Lambda Exo Reaction Buffer (B8030): 1X
DNA: up to 5 µg
Lambda Exonuclease (X8030L): 5 U
Total Volume = 50 µL

Usage Instructions Digestion of 5’ phosphorylated strand of dsDNA.

1. Set up the following reaction mixture in a total volume of 50 µL:

2. Incubate at 37°C for 30 minutes. 3. Reaction can be stopped by adding 10 mM EDTA or incubating at 75°C for 10 min.

Quality Control

Unit activity is measured using a 2-fold serial dilution method. Dilutions of the enzyme were made in 1X reaction buffer and added to 50 µL reactions containing a 1.1 kb tritiated DNA fragment and 1X Lambda Exo Reaction Buffer. Reactions were incubated for 10 minutes at 37°C, plunged on ice, and analyzed using a TCA-precipitation method.

Protein concentration (OD 280 ) is determined by OD 280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample.

Double-stranded endonuclease is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.

E. coli 16S rDNA Contamination is evaluated using 5 µL replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay

Applications

- Plasmid prep cleanup
- Generates single-stranded DNA from linear double-stranded DNA
- Removes oligonucleotide primers after PCR

This product is available for molecular biology applications such as:

References: 1. Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biology (John Wiley and Sons, Inc.) 2. Little, J.W. (1981) Gene Amp. Anal., 2, 135-145.