商品编号


P7460L



品牌


Enzymatics



公司


Enzymatics,inc



公司分类


RNA Enzymes



Size

1,000 U

商品信息

Poly(A) Polymerase


Product Description

Poly(A) Polymerase catalyzes the addition of AMP from ATP to the 3′ hydroxyl of RNA. The reaction requires Mg
2+
?and is template independent.

Source of Protein
The gene encoding?
E. coli
?Poly(A) Polymerase expressed from a plasmid in?
E. coli
.

Supplied in
25 mM Tris-HCl
500 mM NaCl
1mM MgCl
2
0.1 mM DTT
0.1 mM EDTA
50% glycerol
pH 8.0 @ 25°C

Supplied With:
B7460 (10X Poly(A) Polymerase Reaction Buffer)
N2070-10 (10 mM ATP Solution)

10X Poly(A) Polymerase Reaction Buffer (B7460):

500 mM Tris-HCl
2.5 M NaCl
100 mM MgCl
2
pH 7.9 @ 25°C

Unit Definition
1 unit is defined as the amount of enzyme that will incorporate 1 nmol of ATP into acid-insoluble material in 10 minutes at 37°C.



Quality Control Analysis

Unit Characterization Assay
SSpecific activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer (50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl
2
, pH 7.9 @ 25°C, and added to 50 ?L reactions containing a 15-mer RNA Oligo, 1X reaction buffer, 1 mM ATP, 2.5 mM MnCl
2
?and 3H-ATP. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using the method of Sambrook and Russell (
Molecular Cloning, v3, 2001, pp. A8.25-A8.26
).

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 solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 52,060 and molecular weight of 53,870 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 95% 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% 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% 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
Replicate 10 ?L samples were screened for non-specific RNAse contamination using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.


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Product Information



Poly(A) Polymerase


Part Number
P7460L
Price
$363
Concentration
5,000 U/mL
Unit Size
1,000 U


SDS

Available on request








Product Specification*


Storage Temperature
-15 to -25°C


Test

Specification


Purity (SDS-PAGE)
>95%
Specific Activity
>20,000 U/mg
SS Exonuclease
200 U <5.0% released
DS Exonuclease
200 U <1.0% released
DS Endonuclease
200 U = no conversion
E.coli
DNA Contamination
100 U <10 copies
RNAse Contamination
200 U = 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.