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 at 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.

This enzme is supplied in 25 mM Tris-HCl, 50 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA and 50% glycerol: pH 7.5 at 25°C.

10X Lambda Exo Reaction Buffer (cat. no B8030) contains 670 mM glycine and 25 mM MgCl₂, pH 9.4 at 25°C.

SDS available upon request.

Enzyme properties

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

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

Unit definition: 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.

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.

Quality control analysis

Unit activity was measured using a twofold serial dilution method. Dilutions of 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 10 minutes at 37°C, placed on ice, and analyzed using a TCA-precipitation method.

Protein concentration 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 mass of the band corresponding to the protein of interest in the diluted sample.

Double-stranded endonuclease activity 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 contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

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