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ProtoScript® II Reverse Transcriptase
NEBU recombinant 42 65 Heat

Product Introduction

  • Recombinant M-MuLV reverse transcriptase
  • Reduced RNase H activity
  • Increased thermostability
Catalog # Size Concentration
M0368S 4000.0 units 200000 units/ml
M0368L 10000.0 units 200000 units/ml
M0368X 40000.0 units 200000 units/ml

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Product Information

Description

ProtoScript® II Reverse Transcriptase is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild type M-MuLV. The enzyme is active up to 48°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product up to 12 kb.

Protoscript II Reverse Transcriptase performs as well as other RNase H– Reverse Transcriptases Protoscript II Reverse Transcriptase performs as well as other RNase H– Reverse Transcriptases

Jurkat total RNA (1 μg) was used in a 20 μl first strand cDNA synthesis. Mixtures of all reaction components, except for reverse transcriptase, were held at different temperatures for 3 min. 200 units SuperScript® II (A) or NEB’s ProtoScript II Reverse Transcriptase (B) was added and incubated at the indicated temperature for 50 minutes, followed by heat inactivation for 5 min at 80°C. 1 μl of cDNA was used in a 25 μl PCR using LongAmp Taq Master Mix (NEB #M0533) for 35–40 cycles. Ladder L is the 2 Log DNA Ladder (NEB #N0469). 
Robust cDNA synthesis is achieved even with longer templates Robust cDNA synthesis is achieved even with longer templates

Jurkat total RNA (1 μg) was used in a 20 μl first strand cDNA synthesis with 200 units of NEB ProtoScript II Reverse Transcriptase. Reactions were incubated at 42°C for 50 minutes, followed by heat inactivation for 5 minutes at 80°C. 1 μl of cDNA was used in a 25 μl PCR using LongAmp Taq Master Mix (NEB #M0533) for 35–40 cycles. Sizes are indicated above gel. 
Generate high quality cDNA even with very low amounts of starting RNA Generate high quality cDNA even with very low amounts of starting RNA

Decreasing amounts of Jurkat total RNA (1 μg – 1 pg) were used in 20 μl first strand cDNA synthesis with 200 units of NEB ProtoScript II Reverse Transcriptase. Reactions were incubated at 42°C for 50 minutes, followed by heat inactivation for 5 minutes at 80°C. 1 μl of cDNA was used in a 25 μl PCR using LongAmp Taq Master Mix (NEB #M0533) for 40 cycles. The target is a 0.6 kb fragment of GAPDH. Ladder L is the 2-Log DNA Ladder (NEB #N0469). 
ProtoScript II Reverse Transcriptase displays superior sensitivity ProtoScript II Reverse Transcriptase displays superior sensitivity

Decreasing amounts of luciferase mRNA (109 to 102) molecules were converted into cDNA in the presence of 1 ng Jurkat total RNA using 50 units of NEB ProtoScript II Reverse Transcriptase in a total reaction volume of 20 μl. 1/20 of the cDNA product was amplified using SsoAdvanced™ SYBR® Green Supermix. As few as 5 molecules of luciferase mRNA are detectable.

Product Source

The gene encoding a mutant M-MuLV Reverse Transcriptase (RNase H–) is expressed in E. coli and purified to near homogeneity.
This product is related to the following categories:
RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products,
This product can be used in the following applications:
Helicase-dependent Amplification,
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis),
RNA Cloning,
RT-PCR & cDNA Synthesis,
PCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0368S     -20    
      ProtoScript® II Reverse Transcriptase M0368SVIAL -20 1 x 0.02 ml 200,000 units/ml
      ProtoScript® II Reverse Transcriptase Reaction Buffer B0368SVIAL -20 1 x 1.5 ml 5 X
      DTT B1034AVIAL -20 1 x 0.5 ml 0.1 M
  • M0368L     -20    
      ProtoScript® II Reverse Transcriptase M0368LVIAL -20 1 x 0.05 ml 200,000 units/ml
      ProtoScript® II Reverse Transcriptase Reaction Buffer B0368SVIAL -20 1 x 1.5 ml 5 X
      DTT B1034AVIAL -20 1 x 0.5 ml 0.1 M
  • M0368X     -20    
      ProtoScript® II Reverse Transcriptase M0368LVIAL -20 4 x 0.05 ml 200,000 units/ml
      DTT B1034AVIAL -20 4 x 0.5 ml 0.1 M
      ProtoScript® II Reverse Transcriptase Reaction Buffer B0368SVIAL -20 4 x 1.5 ml 5 X

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly(rA)•oligo(dT)18 as template.

Reaction Conditions

1X ProtoScript® II Reverse Transcriptase Reaction Buffer
Incubate at 42°C

1X ProtoScript® II Reverse Transcriptase Reaction Buffer
50 mM Tris-HCl
75 mM KCl
3 mM MgCl2
(pH 8.3 @ 25°C)

Storage Buffer

20 mM Tris-HCl
100 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.01% IGEPAL® CA-630
pH 7.5 @ 25°C

Heat Inactivation

65°C for 20 minutes

Unit Assay Conditions

50 mM Tris-HCl (pH 8.3), 75 mM KCl, 6 mM MgCl2, 10 mM dithiothreitol, 0.01% IGEPAL CA-630, 0.5 mM dTTP, 0.4 mM poly(rA)•oligo(dT)18.

Product Notes

  1. Reaction Conditions:
    1X ProtoScript II Reverse Transcriptase Reaction Buffer, 10 mM DTT, 200 units ProtoScript II Reverse Transcriptase, supplemented with 0.5 mM dNTPs (not included) and 5 µM dT23VN (not included). Incubate at 42°C for 50 minutes. If random primers are used, a 10 minute incubation at room temperature is recommended before transferring to 42°C.

References

  1. Roth, M.J., Tanese, N. and Goff, S.P. (1985). J. Biol. Chem.. 260, 9326-9335.
  2. Kotewicz, M.L. et al, (1988). Nuc. Acids Res.. 16, 265-277.
  3. Lim, D. et al, (2006). J. Virol.. 80, 8379-8389.
  4. Sambrook, J., Fritsch, E.F. and Maniatis, T. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.(Ed.), Molecular Cloning: A Laboratory Manual. 1989, pp. 5.52-5.55, 8.11-8.17.

Protocols, Manuals & Usage

Protocols

  1. First Strand cDNA Synthesis (Quick Protocol) (NEB #M0368)
  2. First Strand cDNA Synthesis (Standard Protocol) (NEB #M0368)
  3. First Strand cDNA Synthesis (No-RT Negative Control Reaction) (NEB #M0368)

Usage & Guidelines

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the difference between NEB# M0368 and NEB# M0253?
  2. What is the optimal reaction temperature for ProtoScript II Reverse Transcriptase (M0368)?
  3. Can the cDNA products be used in real-time PCR analysis?
  4. What thermostable DNA polymerase can be used for PCR after cDNA synthesis?
  5. How can the yield be improved when using ProtoScript II Reverse Transcriptase?
  6. How can the length of the product generated by M-MuLV Reverse Transcriptase be increased?
  7. Is RNaseH treatment required before PCR amplification?
  8. What is the difference between Induro Reverse Transcriptase (NEB #M0681) and ProtoScript II Reverse Transcriptase (NEB #M0368)?
  9. Why do I have low cDNA yields?
  10. How do I know whether my template RNA is of good quality?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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