Lambda Exonuclease

Description:
Lambda Exonuclease is a highly processive exonuclease which selectively degrades the 5′-phosphorylated strand of double-stranded DNA via the stepwise 5′ to 3′ release of mononucleotides from duplex DNA. Lambda Exonuclease is inactive against 5′-hydroxyl termini⁽¹⁾, and will not initiate excision at a nick or gap⁽²⁾, though it will degrade a 5′-overhanging tail from duplex DNA at a greatly reduced rate.

Specific Activity:
100,000U/mg

• Catalyzes the removal of 5′ mononucleotides from duplex DNA.

Source:
Purified from a strain of E. coli that overexpresses the exonuclease gene from bacteriophage Lambda.

Supplied in:
25 mM Tris-HCl
50 mM NaCl
1.0 mM Dithiothreitol
0.1% mM EDTA
50% Glycerol
pH 7.5 @ 25°C

Supplied with:
10X Lambda Exo Reaction Buffer

10X Lambda Exo Reaction Buffer :
670 mM Glycine
25 mM MgCl2
pH 9.4 @ 25°C

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.

Nuclease Contamination Tests

Endonuclease Activity:
A 50 μL reaction containing 0.5 μg of pBR322 DNA and 10 μL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli 16S rDNA Contamination Test:
Replicate 5 μL samples of enzyme solution were 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. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.

Quality Control Analysis

Unit Characterization Assay:
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer ([Lambda Exo]f = 1.25-0.01μg/mL) 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, plunged on ice, and analyzed using a TCA-precipitation method.

Protein Concentration (OD280) Measurement:
A 2.0 μL sample of enzyme was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 46,660 and molecular weight of 25,909 Daltons. Acceptance for this assay is +/- 5% of reference sample.

SDS-Page (Physical Purity Assessment):
2.0 μL of enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 μL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.

Recommended Storage Conditions:
-20°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, 135-145