商品编号


L6010L



品牌


Enzymatics



公司


Enzymatics,inc



公司分类


Ultrapure Ligases



Size

900,000 U

商品信息

T3 DNA Ligase


Product Description

T3 DNA Ligase catalyzes the formation of a phosphodiester bond between a 5′ phosphate and a 3′ hydroxyl termini in duplex DNA. The enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA. In the absence of 20-30% PEG 6000, T3 DNA Ligase displays a very low efficiency for blunt-ended ligation. (1) T3 DNA Ligase displays a higher efficiency for joining A/T overhangs than C/G matched ends. (1) T3 DNA Ligase retains 95% of its activity in 1.0 M NaCl or KCl, with an optimal concentration of 300 mM(1).

Source of Protein
A recombinant?
E. coli
?strain carrying the T3 DNA Ligase gene.

Supplied in
20 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
50% glycerol
pH 7.5 @ 25°C

Supplied With:
B1010 (2X Rapid Ligation Buffer)

2X Rapid Ligation Buffer (B1010):

132mM Tris-HCI
20 mM MgCl
2
2 mM DTT
2 mM ATP
15% PEG 6000
pH 7.6 @ 25°C

Unit Definition
1 unit is defined as the amount of T3 DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 23°C.



Quality Control Analysis

Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X Rapid Ligation Buffer and added to 20 ?L reactions containing double stranded DNA fragments and 1X Rapid Ligation Buffer. Reactions were incubated 30 minutes at 23°C (room temp), plunged on ice, and analyzed on a 1% agarose gel stained with ethidium bromide.

Protein Concentration (OD
280
) Measurement
A 2.0 ?L sample of enzyme was analyzed at OD
280
?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 60,880 and molecular weight of 39,350 Daltons.

SDS-Page (Physical Purity Assessment) and Specifications
2.0 ?L of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW
Marker
and 2.0 ?L 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.



Contamination Tests

Single-Stranded Exonuclease Activity
A 50 ?L reaction containing 10,000 cpm of a r
ADI
olabeled single-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity
A 50 ?L reaction containing 5,000 cpm of a r
ADI
olabeled double-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts

Double-Stranded Endonuclease Activity
A 50 ?L reaction containing 0.5 ?g of pBR322 DNA and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in no visually discern
IBL
e conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli
?16S rDNA Contamination Test
Replicate 5uL 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 (C
t
) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control C
t
?values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.


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Product Information



T3 DNA Ligase


Part Number
L6010L
Price
$363
Concentration
3,000,000 U/ml
Unit Size
900,000 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>99%
Specific Activity
3,000,000 U/mg
SS Exonuclease
30,000 U <1.0% released
DS Exonuclease
30,000 U <1.0% released
DS Endonuclease
30,000 U = No conversion
E.coli
DNA Contamination
30,000 U <10 copies





* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.

Notes

T3 DNA Ligase is supplied with 2X Rapid Ligation Buffer for use in ligation reactions.

The enzyme has also been characterized in the following buffer, with a 17-fold decrease in specific activity due to the absence of the crowding agent (PEG 6000):

1X T3 DNA Ligase Buffer:
50 mM Tris-HCl
300 mM NaCl
0.5 mM ATP
1 mM DTT
pH 8.0 @ 25°C

The following information was taken from the Cai reference (cited below), which describes the characterization of the T3 DNA Ligase:

Optimal pH: 8.0
Optimal reaction temperature: 20°C
Optimal reaction time: >30 minutes (without PEG 6000)
Optimal ionic strength: 300 mM NaCl
Optimal divalent cation/concentration: 2 mM Mg++
Optimal cofactor: ATP (0.5 mM)

References


Cai, Liang, et al. (2004) J. Biochem. 135, 397-403




Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for admi
NIST
ration to humans or animals. SDS sheets relevant to this product are available upon request.