Lambda Exonuclease

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

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 MgCl₂, pH 9.4 at 25°C.

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

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.

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 (OD₂₈₀) is determined by OD₂₈₀ 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

This product is available for molecular biology applications such as:

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

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.