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商品編號


G5020L



品牌


Enzymatics



公司


Enzymatics,inc



公司分類(lèi)


DNA Modifying Enzymes



Size

500 U

商品信息

Thermo
l
ABI
le UDG


Product Description

Thermo
l
ABI
le Uracil-DNA Glycosylase removes uracil from DNA by hydrolyzing the N-glycosylic bond between the deoxyribose and the base leaving an AP (apurinic or apyrimidinic) site. This enzyme ( 1-10 units) is completely inactivated by a 10 minute incubation at temperatures greater than 50°C in the 1x reaction buffer as measured in the unit characterization assay.

Source of Protein
A recombinant?
E. coli
?strain carrying the recombinant
Thermo
l
ABI
le Uracil DNA Glycosylase gene isolated from a marine bacteria.

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

Supplied With:
B5020 (10X Reaction Buffer)

10X Reaction Buffer (B5020):

700 mM Tris-HCl
100 mM NaCl
10 mM EDTA
1 mg/mL BSA
pH 8.0 @ 25°C

Unit Definition
1 unit is defined as the amount of
Thermo
l
ABI
le UDG required to release 1nmol of Uracil from dU-containing DNA in one hour at 37°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 reaction buffer (70mM Tris-HCl, 10mM NaCl, 1mM EDTA, 100 ?g/mL BSA, pH 8.0 @ 25°C) and added to reactions containing a?
3
H-dUTP labeled 1.1kb PCR product in 1X reaction buffer. Reactions were incubated for 60 minutes at 37°C, plunged on ice, and analyzed using a TCA-precipitation method.

Protein Concentration (OD
280
) Measurement
A 2.0 ?L sample of enzyme was analyzed at OD
280
?using a
Nanodrop
ND-2000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (
Pierce
Cat #23209), and blanked with product storage buffer. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 41,820 and molecular weight of 26,218 Daltons.

SDS-Page (Physical Purity Assessment)
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 same enzyme species. 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 98% 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 5 ?L 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



Thermo
l
ABI
le UDG


Part Number
G5020L
Price
$363
Concentration
1,000 U/ml
Unit Size
500 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>98%
Specific Activity
30,000 U/mg
SS Exonuclease
10 Units <1.0% released
DS Exonuclease
10 Units <1.0% released
DS Endonuclease
5 Units = No conversion
E.coli
DNA Contamination
5 Units < 10 copies





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

Notes

Thermo
l
ABI
lity was examined by heat treating 10 Units of enzyme for 10 minutes at 50°C in 1X reaction buffer. The enzyme was transferred to reactions containing a
3
H-dUTP 1.1kb PCR product and activity was measured as described in the unit characterization assay. After heat treatment, less than 0.5% activity remains.



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.