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商品編號


Y9220L



品牌


Enzymatics



公司


Enzymatics,inc



公司分類(lèi)


RNA Enzymes



Size

5,000 U

商品信息

RNAse H


Product Description

E.coli
?RNase H (rnh) is an endoribonuclease which degrades the RNA strand of RNA/DNA hybrid molecules. RNase H digestion produces ribonucleotide molecules with 5-phosphate and 3-hydroxyl termini. RNAse H is nearly inactive against single or double-stranded RNA molecules.

Source of Protein
A recombinant?
E. coli
?strain carrying the RNAse H (rnh) gene from?
E.coli.

Supplied in
20 mM Tris-HCl
100 mM KCl
10 mM MgCl
2
0.1 mM EDTA
0.1 mM DTT
50% glycerol
pH 7.9 @ 25°C

Supplied With?
B9220 (10X RNAse H Buffer)

10X RNAse H Buffer (B9220):
500 mM Tris-HCl
750 mM KCl
30 mM MgCl
2
100 mM DTT
pH 8.3 @ 25°C

Unit Definition
1 unit is defined as the amount of enzyme that will hydrolyze 1 nmol of RNA from an?
3
H-labeled DNA:RNA hybrid molecule into acid-soluble material in 20 minutes at 37°C.



Quality Control Analysis

Unit Characterization Assay
Specific activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X RNAse H reaction buffer and added to 50 ?L reactions containing?
3
H-labeled poly(rA), poly (dT) DNA, and 1X RNAse H Buffer. Reactions were incubated 20 minutes at 37°C, plunged on ice, and release of TCA soluble counts was analyzed.

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 40,540 and molecular weight of 18,053 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 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 5.0% 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.

Non-Specific RNAse Assay
Product was screened for non-specific RNAse contamination using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.


View PDF Poster Instructions FAQ


Product Information



RNAse H


Part Number
Y9220L
Price
$363
Concentration
5,000 U/ml
Unit Size
5,000 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>99%
Specific Activity
625,000 U/mg
SS Exonuclease
500 U <5.0% released
DS Exonuclease
500 U <1.0% released
DS Endonuclease
500 U = No conversion
E.coli
DNA Contamination
500 U <10 copies
RNAse Contamination
500 = No Detectable non-specific RNase





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



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.