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商品編號


P7600L



品牌


Enzymatics



公司


Enzymatics,inc



公司分類(lèi)


New Products



Size

10,000 U

商品信息

EnzScript? (MMLV Reverse Transcriptase RNase H-)


Product Description

EnzScript? (M-MLV Reverse Transcriptase RNase H minus) is an RNA-dependent DNA polymerase with no detectable RNase H activity. EnzScript? can be used to generate first-strand
CDN
A from polyA mRNA or total RNA for use in downstream applications such as RT- PCR,
CDN
A cloning or library construction for RNA-Seq. Point mutations in the RNase H domain increase the
Thermo
st
ABI
lity of the enzyme and support greater
CDN
A yield of full-length transcripts than wild type M-MLV Reverse Transcriptase (1).

Source of Protein:
A recombinant E. coli strain carrying the Moloney-Murine Leukemia Virus Reverse Transcriptase gene with 3 point mutations in the RNase H domain that eliminate detectable RNase H activity.

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.

Molecular weight:
75,938 Daltons



Quality Control Analysis

Unit Activity
?is measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X M-MuLV RT RNAse H- 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).

Reverse Transcriptase function
?was determined by the enzyme’s
ABI
lity to generate a 9.4 kB
CDN
A transcript. Following 2-Step RT-PCR the 9.4Kb amplicon was visualized by agarose gel electrophoresis

Protein Concentration (OD
280
)
?is determined by OD
280
?absorbance. Physical Purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.

Single-Stranded Exonuclease
?is determined in a 50 ?L reaction containing a r
ADI
olabeled single-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C.

Double-Stranded Exonuclease
?is determined in a 50 ?l reaction containing a r
ADI
olabeled double-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C.

Double-Stranded Endonuclease
?is determined in a 50 ?L reaction containing 0.5 ?g of plasmid DNA and 10 ?L of enzyme solution incubated for 4 hours at 37°C.

E.coli
?16S rDNA Contamination
?is evaluated using 5 ?L replicate samples of enzyme solution 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.

Non-Specific RNAse
?contamination is assessed using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

Supplied in:
?20 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA , 1 mM DTT, 0.01% NP-40 Alternative, 50% glycerol, pH 7.5 @ 25°C.

Supplied with:
?5X M-MLV Reverse Transcriptase RNAse H- Reaction Buffer (B7601) and 100mM DTT (B9060)

Kit Contents




Part

Part Number



EnzScript?

P7600



5X M-MLV Reverse Transcriptase RNase H- Buffer

B7601



100 mM DTT

B9060




Polymerase Properties

Molecular Weight: 75.9 kDa
Optimum Extension Temperature: 42°C
RNase H activity: none detected
Unit Concentration: 200 U/?l
Transcript Length: 12.3 kB

Common Applications

EnzScript? (M-MLV Reverse Transcriptase RNase H minus) is an RNA- dependent DNA polymerase commonly used to synthesize First- Strand
CDN
A for RT-PCR amplification,
CDN
A cloning or RNASeq. Reduced RNase H activity enables greater yield of full-length
CDN
A transcripts (> 5 kb) and increased thermal st
ABI
lity over standard M-MLV RT.

First Strand Reaction Protocol

General precaution against RNase degradation of template RNA should be taken when setting up First-Strand reactions such as use of nuclease-free water, RNase inhibitor, RNase-free tubes and sterile pipet tips with filters. The following procedure can be used as a guideline for preparing a 20 ?l First-Strand
CDN
A reaction.

Materials to be provided by User:

? Sterile, nuclease-free water
? Primer (oligo dT
(15-20)
?
or
?random hexamers?
or?
gene-specific)
? dNTP mix (dATP, dCTP, dGTP, dTTP)
? RNA template
? RNase Inhibitor

1. Add the following components to an RNase-free microcentrifuge tube on ice. For more than one reaction, prepare a mastermix.




Component

Volume



50 ?M oligo dT(15-20)
or
50 ?M random hexamers
or
10 ?M gene-specific primer

1 to 2 ?l



5 mM dNTP mix

2 ?l



RNA template*

X ?l



Sterile, nuclease-free water

to 12 ?l




* 1 ng to 1 ?g total RNA?
or
?1 to 250 ng mRNA

2. Heat microcentrifuge tube to 65°C for 5 minutes and quickly cool on ice for 2 minutes to anneal primer to RNA template. Spin tube briefly to collect condensate.

3. Add the following components (to each First-Strand reaction) to the microcentrifuge tube on ice:




Component

Volume



5X M-MLV Reverse Transcriptase
RNase H- Buffer

4 ?l



100 mM DTT

2 ?l



RNase Inhibitor (optional) or
nuclease-free water

1 ?l



200 U EnzScript?

1 ?l




4. Incubate each 20 ?l First Strand reaction as follows:


25°C for 2 minutes (oligo dT(15-20), gene-specific primer)?or?25°C for 10 minutes (random hexamer)

42°C for 30 to 60 minutes

Heat kill at 70°C for 15 minutes


5. Use
CDN
A in downstream application or store at -20°C. For RT- PCR, 1 to 2 ?l of
CDN
A from First-Strand reaction is typically added as template to PCR. Optional: Remove RNA strand prior to PCR by adding 1 ?l RNase H (5 U) to
CDN
A:RNA hybrid, incubate at 37°C for 20 minutes and 65°C for 10 minutes (heat kill). RNase H treatment is recommended for amplification of long amplicons (> 5 kB).

Related
Enzymatics
Products




Part

Part Number



25 mM dNTP mix

N2050



RNase Inhibitor

Y9220



RNase H

Y9240




Frequently Asked Questions and Troubleshooting

For Frequently Asked Questions (FAQ) and troubleshooting please click here.


View PDF Poster Instructions FAQ


Product Information



EnzScript? (MMLV Reverse Transcriptase RNase H-)


Part Number
P7600L
Price
$128/pack
Concentration
200,000 U/mL
Unit Size
10,000 U
Lot Number
Shipment dependent


SDS

Available on request








Product Specification*


Storage Temperature
-25°C to -15°C


Test

Units Tested

Specification


SDS Purity
n/a
> 99%
Specific Activity
n/a
≥ 280,000 U/mg
SS Exonuclease
2,000
< 5.0%
DS Exonuclease
2,000
< 1.0%
DS Endonuclease
2,000
No Conversion
E.coli
DNA Contamination
2,000
< 10 copies
RNAse Contamination
2,000
No Detectable non-specific RNase
Functional RT-PCR Assay
n/a
Synthesis of 9.4 kb
CDN
A transcript





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

Additional Downloads

Product Note

References


Gerard G.F. et al.,
Nucl. Acids Res.
(2002) 30 (14): 3118-3129




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.