PRODUCT DESCRIPTION产品描述
EnzScript? (M-MLV Reverse Transcriptase RNase H minus) is an RNA-dependent DNApolymerase with no detectable RNase H activity. EnzScript? can be used to generatefirst-strand cDNA from polyA mRNA or total RNA for use in downstreamapplications such as RT- PCR, cDNA cloning or library construction for RNA-Seq.Point mutations in the RNase H domain increase the thermostability of theenzyme and support greater cDNA yield of full-length transcripts than wild typeM-MLV Reverse Transcriptase (1).
Source of Protein蛋白来源
A recombinant E. coli strain carrying theMoloney-Murine Leukemia Virus Reverse Transcriptase gene with 3 point mutationsin the RNase H domain that eliminate detectable RNase H activity.
Unit Definition单位定义
1 unit is defined as the amount of enzymerequired to incorporate 1 nmol of dTTP into acid insoluble material in 10minutes at 37°C using polyr(A)/oligo (dT) as a substrate.
Molecular weight: 75,938 Daltons
PRODUCT INFORMATION产品信息
EnzScript? (MMLV Reverse Transcriptase RNase H-)
Part Number P7600L
Concentration 200,000 U/mL
Unit Size 10,000 U
Lot Number Shipmentdependent
SDS Available on request
PRODUCT SPECIFICATION*产品特性
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 cDNA transcript
COMMON APPLICATIONS常见应用
EnzScript? (M-MLV Reverse Transcriptase RNase H minus) is an RNA- dependentDNA polymerase commonly used to synthesize First- Strand cDNA for RT-PCRamplification, cDNA cloning or RNASeq. Reduced RNase H activity enables greateryield of full-length cDNA transcripts (> 5 kb) and increased thermalstability over standard M-MLV RT.
FIRST STRAND REACTION PROTOCOL
General precaution against RNasedegradation of template RNA should be taken when setting up First-Strandreactions such as use of nuclease-free water, RNase inhibitor, RNase-free tubesand sterile pipet tips with filters. The following procedure can be used as aguideline for preparing a 20 μl First-Strand cDNA reaction.
MATERIALS TO BE PROVIDED BY USER:用户自备
? Sterile,nuclease-free water
? Primer(oligo dT(15-20) or random hexamers or gene-specific)
? dNTP mix(dATP, dCTP, dGTP, dTTP)
? RNA template
? RNaseInhibitor
1. Add the following components to anRNase-free microcentrifuge tube on ice. For more than one reaction, prepare amastermix.
Component Volume
50 μM oligo dT(15-20) or
50 μM random hexamers or 1 to 2 μl
10 μM gene-specific primer
5 mM dNTP mix 2 μl
RNA template* X μl
Sterile, nuclease-free water to 12 μl
* 1 ng to 1 μg total RNA or 1 to 250 ngmRNA
2. Heat microcentrifuge tube to 65°C for 5 minutes and quickly cool onice for 2 minutes to anneal primer to RNA template. Spin tube briefly tocollect condensate.
3. Add the following components (to eachFirst-Strand reaction) to the microcentrifuge tube on ice:
Component Volume
5X M-MLV Reverse
Transcriptase 4 μl
RNase H- Buffer
100 mM DTT 2 μl
RNase Inhibitor
(optional) or 1 μl
nuclease-free water
200 U EnzScript? 1 μl
4. Incubate each 20 μl First Strandreaction 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 cDNA indownstream application or store at -20°C. For RT- PCR, 1 to 2 μl of cDNA from First-Strand reaction istypically added as template to PCR. Optional: Remove RNA strand prior to PCR byadding 1 μl RNase H (5 U) to cDNA:RNA hybrid, incubate at 37°C for 20 minutes and 65°C for 10 minutes (heat kill). RNaseH treatment is recommended for amplification of long amplicons (> 5 kB).
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