商品编号


P7040L



品牌


Enzymatics



公司


Enzymatics,inc



公司分类


Ultrapure Polymerases



Size

100,000 U

商品信息

M-MuLV Reverse Transcriptase


Product Description

M-MuLV Reverse Transcriptase is a DNA polymerase which utilizes RNA as a substrate and exhibits no measurable proofre
ADI
ng 3′→5′ exonuclease function.? This enzyme can perform
CDN
A synthesis by extending off a DNA primer annealed to an RNA template, or can copy a single-stranded DNA template.

Source of Protein

A recombinant?
E. coli
?strain carrying the Moloney-Murine Leukemia Virus Reverse Transcriptase gene.

Supplied in

50 mM Tris-HCl
150 mM NaCl
0.1 mM EDTA
1 mM DTT
0.1% NP-40 Alternative
50% glycerol
pH 7.6 @ 25°C

Supplied With
B7040 (10X M MuLV RT Buffer)

10X M-MuLV RT Buffer (B7040):
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 required to incorporate 1 nmol of dTTP into acid insoluble material in 10 minutes at 37°C using poly r(A)/oligo (dT) as a substrate.

?



Quality Control Analysis

Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X M-MuLV RT Buffer and added to 50 ?L reactions containing 20 ?g/mL poly r(A) RNA, oligo (dT) DNA, 1X RT Buffer, 3H-dTTP and 250 ?M dTTP. 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
Protein concentration is determined utilizing
Bio-Rad
Protein Assay Kit II (500-0002). Three replicate 5.0uL samples of enzyme were tested at concentrate, 1:10 and 1:100 dilutions. Absorbance at 595nm and comparison to a BSA standard curve in the 1-10ug range allows for relative protein determination.

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 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.

Non-Specific RNAse Assay
Replicate 10 uL samples were 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



M-MuLV Reverse Transcriptase


Part Number
P7040L
Price
$363
Concentration
200,000 U/ml
Unit Size
100,000 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>99%
Specific Activity
169,000 U/mg
SS Exonuclease
2,000 U < 5.0% released
DS Exonuclease
2,000 U < 1.0% released
DS Endonuclease
2,000 U = No conversion
E.coli
DNA Contamination
2,000 U < 10 copies
RNAse Contamination
2,000 U = No Detectable non-specific RNAse





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

Usage Instructions

First Strand Synthesis


Primer Annealing:
Combine the following in an RNase-free reaction vessel:





Amount

Description

Final Concentration



1 ?L

25mM dNTP Solution (N2050L)

2.0 mM



X ?L

1ng-2?g Total RNA

-or-





X ?L

5-500 ng mRNA (polyA selected)




1 ?L

Oligo (dT)
12-18
(500 ?g/ml)

-or-


40 ?g/mL



1 ?L

Random Primers (125 ?g/ml)

-or


10 ?g/mL



1 ?L

GSP Primer (2 pmol)

165 ?M



X ?L

Sterile, Type I Water

N/A



10 ?L

Total Volume




?


Heat reaction for 5 minutes at 65°C. Spin briefly (5 sec) to pull down condensate and place immediately on ice.

Add 1 ?L 10X M-MuLV RT Buffer (B7040) and Type I Water to a final volume of 10 ?L per reaction.

Incubate:

If using Oligo (dT) or GSP primers: 2 minutes @ 42°C

If using Random primers: 2 minutes @ 25°C



Add 1 ?L (200 units) M-MuLV Reverse Transcriptase (P7040) and mix by gently pipetting sample. (Note: if using random primers, pre-incubate reaction @25°C for 10 minutes).

Incubate at 42°C for 45-60 minutes.

Inactivate enzyme at 85°C for 10 minutes.

Store products at -20°C or proceed to next step.




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.