EnzScript Reverse Transcriptase

For reverse transcription from RNA for first strand cDNA synthesis and RT-PCR

Products

This product is available worldwide except in China. Kindly note that the delivery takes two to three weeks at the moment. For OEM orders in all countries, including China, please visit our OEM pages.We aim to make this product available globally and speed up delivery. We will keep you posted with any development. Thank you for choosing us as your molecular biology partner.
The EnzScript Reverse Transcriptase is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.
Image
EnzScript Reverse Transcriptase (10,000 U)

Cat. No. / ID:  P7600L

10,000 U of EnzScript Reverse Transcriptase (0.05 mL at 200,000 U/mL) and 5x M-MLV Reverse Transcriptase RNase H Minus Buffer (1 x 1.5 mL) and 100 mM DTT (1 x 1.5 mL)
Copy order details
R$1,115.00

Features

  • RNase H-deficient M-MLV with increased thermal stability
  • Suitable for cDNA cloning and RT-PCR
  • Recommended for library construction for RNA-Seq transcriptome studies
  • Compatible with random hexamer, oligo(dT) and gene-specific priming

Product Details

EnzScript Reverse Transcriptase is a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase with point mutations in the RNase H domain that eliminate 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. The 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.


One unit of EnzScript Reverse Transcriptase 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.


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


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

 

Performance

Polymerase properties

  • Optimum extension temperature: 42°C
  • Transcript length: Up to 9.4 kb
Test Specification
Purity >95%
Specific activity 180,000 U/mg
Single-stranded exonuclease 2000 U <5% released
Double-stranded exonuclease 2000 U <1% released
Double-stranded endonuclease 2000 U = No conversion
E. coli DNA contamination 2000 U <10 copies
RNase contamination 2000 U; No detectable non-specific RNase
Functional RT-PCR assay Synthesis of 9.4 kb cDNA transcript

 

Principle

Reverse transcriptase (RT), also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into DNA. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes.


Reverse transcriptases are used in molecular biology to detect and manipulate RNA molecules through amplification. Reverse transcriptases polymerize a strand of DNA (cDNA) that is complimentary to an original RNA template. The cDNA can then be further amplified through PCR, and qPCR for downstream analysis of transcripts and gene expression.


Native retroviral RT has three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H (RNase H) activity and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA.


RNase H activity degrades the RNA strand of a DNA/RNA hybrid and frees the newly made DNA strand for use as a template in the synthesis of the second strand of DNA. RNase H activity is considered disadvantageous for synthesis of long cDNAs due to the potential of the enzyme to degrade the RNA template before completion of full-length reverse transcription.


EnzScript Reverse Transcriptase has no detectable RNase H activity and has increased thermostability. The enzyme is suitable for generating 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.

 

Procedure

Instructions for using EnzScript Reverse Transcriptase are provided in the corresponding kit protocol in the resources below.


Quality Control


Unit activity of EnzScript Reverse Transcriptase was measured using a two-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 and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).


Protein concentration of EnzScript Reverse Transcriptase (OD280) was determined by OD280 absorbance.


EnzScript Reverse Transcriptase physical purity was evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity was 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 in EnzScript Reverse Transcriptase was 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 in EnzScript Reverse Transcriptase was 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 in EnzScript Reverse Transcriptase enzyme was 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 contamination of EnzScript Reverse Transcriptase was 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 of EnzScript Reverse Transcriptase was assessed using a nuclease detection kit following the manufacturer’s guidelines.


Reverse transcriptase function of EnzScript was determined by the enzyme’s ability to generate a 9.4 kB cDNA transcript. Following 2-Step RT-PCR, the 9.4 Kb amplicon was visualized by agarose gel electrophoresis.

 

Applications

This product is available for molecular biology applications such as:

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

Resources

Protocol Files (1)
Safety Data Sheets (1)

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 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 (RT) RNase H- contains three point mutations that eliminate any measureable RNase H activity, which is native to wild type M-MLV RT. Loss of RNase H activity enables greater yield of full-length cDNA transcripts ( > 5kb) and increased thermal stability over wild type M-MLV RT*. Increased thermostability allows for higher incubation temperatures of the first-strand reaction (up to 50°C), aiding in denaturation of template RNA secondary structure or GC-rich regions.

*Reference:

Gerard G.F. et al., Nucl. Acids Res. (2002) 30 (14): 3118-3129
FAQ ID - 3960
Is this available without glycerol?
Yes. Please contact your business development manager for a custom formulation.
FAQ ID - 3959
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 types of priming are compatible with EnzScript M-MLV RT RNase H-?
EnzScript M-MLV RT RNase H- is compatible with random hexamer priming, poly oligo(dT)/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) / anchored poly oligo(dT) is a more specific priming method where the primer hybridizes to the polyA 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 RNA input amount is low (< 100 ng).
FAQ ID - 3961
What PCR enzymes can be used following 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 (> 2 kB, up to 12 kB), we suggest VeraSeq® Ultra DNA Polymerase (ENZ P7520). 

Note: Generally, 1 to 2 µl (5 to 10%) of cDNA from the first-strand reaction is added as template to a 50 µl PCR reaction, 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*.

*Reference:

Kitabayashi M. et al., Biosci. Biotechnol. Biochem. (2003) 67 (11):2474-6

FAQ ID - 3963
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 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 to 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 (2).  In a two-step RT PCR application, the cDNA added as template into the PCR reaction is generally 1 to 2 µl (5 to 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 - 3964
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