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 cDNA from polyA mRNA or total RNA for use in downstream applications such as RT- PCR, cDNA cloning or library construction for RNA-Seq. Point mutations in the RNase H domain increase the thermostability of the enzyme and support greater cDNA 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
|Storage Temperature||-25°C to -15°C|
|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|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
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 µL reactions containing 20 µg/mL poly r(A) RNA, oligo (dT) DNA, 1X RT Buffer, 3H-dTTP and 250 µM 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 ability to generate a 9.4 kB cDNA transcript. Following 2-Step RT-PCR the 9.4Kb amplicon was visualized by agarose gel electrophoresis
Protein Concentration (OD280) is determined by OD280 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 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.
Double-Stranded Exonuclease is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.
Double-Stranded Endonuclease is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.
E.coli 16S rDNA Contamination is evaluated using 5 µL 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)
|5X M-MLV Reverse Transcriptase RNase H- Buffer||B7601|
|100 mM DTT||B9060|
Molecular Weight: 75.9 kDa
Optimum Extension Temperature: 42°C
RNase H activity: none detected
Unit Concentration: 200 U/µl
Transcript Length: 12.3 kB
EnzScript™ (M-MLV Reverse Transcriptase RNase H minus) is an RNA- dependent DNA polymerase commonly used to synthesize First- Strand cDNA for RT-PCR amplification, cDNA cloning or RNASeq. Reduced RNase H activity enables greater yield of full-length cDNA transcripts (> 5 kb) and increased thermal stability 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 µl First-Strand cDNA 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.
|50 µM oligo dT(15-20) or
50 µM random hexamers or
10 µM gene-specific primer
|1 to 2 µl|
|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 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:
|5X M-MLV Reverse Transcriptase
RNase H- Buffer
|100 mM DTT||2 µl|
|RNase Inhibitor (optional) or
|200 U EnzScript™||1 µl|
4. Incubate each 20 µl 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 cDNA in downstream application or store at -20°C. For RT- PCR, 1 to 2 µl of cDNA from First-Strand reaction is typically added as template to PCR. Optional: Remove RNA strand prior to PCR by adding 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). RNase H treatment is recommended for amplification of long amplicons (> 5 kB).
RELATED ENZYMATICS PRODUCTS
|25 mM dNTP mix||N2050|
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.