EnzScript Reverse Transcriptase

For commercial manufacturing of molecular biology products (OEM)

Features

  • RNA-dependent DNA polymerase with no detectable RNase H activity
  • RNase-H-deficient MMLV reverse transcriptase
  • Increased thermal stability compared to M-MLV
S_1143_6_OEM_Generic_Product

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

EnzScript Reverse Transcriptase

Cat. No. / ID: P7600L

10,000 U (evaluation pack) EnzScript Reverse Transcriptase (200,000 U/ml), 5X M-MLV Reverse Transcriptase RNase H– Buffer and 100 mM DTT
The EnzScript Reverse Transcriptase is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

EnzScript Reverse Transcriptase is a MMLV Reverse Transcriptase RNase H–. This RNA-dependent DNA polymerase exhibits no detectable RNase H activity. EnzScript can be used to generate first-strand cDNA from poly-A 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 (Gerard, G. F.. et al. (2002) Nuc. Acids Res. 30:3118).

EnzScript Reverse Transcriptase is supplied in 20 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA , 1 mM DTT, <0.01% NP-40 Alternative and 50% glycerol; pH 7.5 at 25°C.

The enzyme is supplied with 5X M-MLV Reverse Transcriptase RNase H– Reaction Buffer (cat. no. B7601) and 100mM DTT (cat. no. B9060).

Performance

OEM by QIAGEN offers bulk manufacturing of EnzScript Reverse Transcriptase in custom formulations.

Product properties
Storage temperature: –25°C to –15°C
Optimum extension temperature: 42°C
Transcript length: 12.3 kB

Test Units tested Specification
SDS purity n/a >95%
Specific activity n/a 180,000 U/mg
Single-stranded exonuclease 2000 <5.0%
Double-stranded exonuclease 2000 <1.0%
Double-stranded endonuclease 2000 No conversion
E. coli DNA contamination 2000 <10 copies
RNase contamination 2000 No detectable non specific RNase
Functional RT-PCR assay n/a Synthesis of 9.4 kb cDNA transcript

Principle

The source of the protein is a recombinant E. coli strain carrying the Moloney-Murine Leukemia Virus Reverse Transcriptase gene with three point mutations in the RNase H domain that eliminate detectable RNase H activity

One 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.

The molecular weight of the protein is 75,938 Daltons.

Procedure

First-strand reaction protocol

General precaution against RNase degradation of template RNA should be taken when setting up first-strand reactions. Such precautions include 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 not included:
• Sterile, nuclease-free water
• Primer (oligo dT(15-20) or random hexamers or gene-specific)
• dNTP mix (dATP, dCTP, dGTP, dTTP, cat. no. N2050L)
• RNA template
• RNase Inhibitor (cat. no. Y9240L)
• RNase H (cat. no. Y9240L)

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

Component Volume
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: 1 ng to 1 µg total RNA or 1 to 250 ng mRNA Variable
Sterile, nuclease-free water To total volume of 12 µl

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 µl
100 mM DTT 2 µl
RNase Inhibitor (optional) or
nuclease-free water
1 µl
200 U EnzScript Reverse Transcriptase 1 µl

4. Incubate reactions 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 30–60 minutes
• Heat kill at 70°C for 15 minutes

5. Use cDNA in downstream application or store at –20°C. For RT-PCR, 1–2 µl cDNA from the first-strand reaction is typically added as template to PCR.

Optional: Remove RNA strand prior to PCR by adding 1 µl RNase H (cat. no. Y9220L) 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).

QUALITY CONTROL ANALYSIS

Unit activity is measured using a twofold 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, placed on ice and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, A8.25–A8.26).

Reverse transcriptase function is determined by the enzyme’s ability to generate a 9.4 kB cDNA transcript. Following two-step RT-PCR, the 9.4 kb 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 activity 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 activity 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 activity 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.

FREQUENTLY ASKED QUESTIONS AND TROUBLESHOOTING
For Frequently Asked Questions (FAQ) and troubleshooting please click here .

Applications

• RT-PCR amplification
• cDNA cloning
• Library construction for RNA-seq

FAQ

What is the suggested protocol for generating long, full-length cDNA transcripts (>5 kb)?
EnzScript first-strand reactions containing up to 1 µg total RNA primed with a GSP or poly oligo(dT) and incubated at 42–50°C for up to 60 minutes is recommended for generating long (>5 kb) transcripts. In general, RNase H (ENZ Y9220) treatment of cDNA reactions (5 U, incubation at 37°C for 20 minutes, heat kill at 65°C for 10 minutes) prior to PCR is recommended when amplification of long transcripts is desired (Kitabayashi, M., et al. (2003) Biosci. Biotechnol. Biochem. 67:2474). In a two-step reverse transcription PCR application, the cDNA added as template for the PCR is generally 1–2 µl (5–10%) of the first-strand reaction. VeraSeq Ultra DNA Polymerase (ENZ P7520L) is recommended for PCR when amplifying long transcripts (up to 12 kb).






FAQ ID - 3842
What is the optimal reaction temperature for EnzScript M-MLV Reverse Transcriptase RNase H-?
For routine first-stand synthesis, incubation at 42°C is recommended. For RNA templates with difficult regions containing secondary structures or GC-rich regions, incubation temperatures up to 50°C may be used to increase accessibility of EnzScript M-MLV Reverse Transcriptase RNase H– to these regions.

Note: A low temperature incubation step of 25°C for 2 minutes (poly oligo(dT)) or 25°C for 10 minutes (random hexamers) is recommended for primer extension (to increase the primer Tm) before elevating the temperature for first-strand synthesis. 




FAQ ID - 3840
What are the advantages of EnzScript M-MLV Reverse Transcriptase RNase H-(P7600) versus wild type M-MLV Reverse Transcriptase (P7040)?
EnzScript M-MLV Reverse Transcriptase RNase H contains three point mutations that eliminate any measurable RNase H activity, an activity that is native to wild type M-MLV Reverse Transcriptase. Loss of RNase H activity enables greater yield of full length cDNA transcripts (>5kb) and increased thermal stability over wild type M-MLV Reverse Transcriptase (Gerard, G. F., et al. (2002) Nucl. Acids Res. 30:3118). Increased thermostability allows higher incubation temperatures of the first-strand reaction (up to 50°C), aiding in denaturation of template RNA secondary structure or GC-rich regions.


FAQ ID - 3838
What PCR enzymes can be used following the first-strand synthesis?
For robust PCR amplification of targets (2 kb or less) among the cDNA pool generated in first-strand synthesis, Phoenix Hot Start Taq DNA Polymerase (ENZ P7590) is recommended. For amplification of long PCR targets (from >2 kb to 12 kb), we suggest VeraSeq Ultra DNA Polymerase (ENZ P7520). 

Note: Generally, 1–2 µl (5–10%) of cDNA from the first-strand reaction is added as template to a 50 µl PCR, although the precise amount of cDNA added may require optimization depending on target abundance. Addition of RNase H (ENZ Y9220) (5 U, incubation at 37°C for 20 minutes, heat kill at 65°C for 10 minutes) to degrade the RNA strand following cDNA synthesis (prior to PCR addition) may increase PCR amplification efficiency for some targets, especially those >1 kb (Kitabayashi, M., et al. (2003) Biosci. Biotechnol. Biochem. 67:2474).




FAQ ID - 3841
What types of priming are compatible with EnzScript M-MLV Reverse Transcriptase RNase-H-?
EnzScript M-MLV Reverse Transcriptase RNase H is compatible with random hexamer priming, poly oligo(dT) or anchored poly oligo(dT) priming and gene-specific priming (GSP). Random hexamer priming is the most non-specific priming method and is commonly used for full coverage of fragmented RNA in library construction protocols. Poly oligo(dT) and anchored poly oligo(dT) are more specific priming methods, in which the primer hybridizes to the poly-A tail of mRNA found in eukaryotes. GSP is the most specific priming method, in which the primer hybridizes to a specific target or gene of interest. As a general precaution against RNase degradation of the RNA template, inclusion of an RNase Inhibitor (ENZ Y9240) is suggested especially when the RNA input amount is low (<100 ng).



FAQ ID - 3839