Product Class: Kit

OneTaq® One-Step RT-PCR Kit

Product Introduction

Easy and efficient One-Step RT-PCR

  • Optimized reaction mix for cDNA synthesis and PCR amplification in one step
  • Hot start formulation eliminates nonspecific amplification and does not require a separate activation step
  • Offered with Quick-Load® dyes for sample tracking and direct loading of PCR products onto gels
Catalog # Size Concentration
E5315S 30.0 reactions

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Product Information

Description

The OneTaq One-Step RT-PCR Kit offers sensitive and robust end-point detection of RNA templates. cDNA synthesis and PCR amplification steps are performed in a single reaction using gene-specific primers, resulting in a streamlined RT-PCR protocol.

The kit combines three optimized mixes: OneTaq One-Step Enzyme Mix, OneTaq One-Step Reaction Mix and OneTaq One-Step Quick-Load Reaction Mix. The Enzyme Mix combines ProtoScript II Reverse Transcriptase, Murine RNase Inhibitor and OneTaq Hot Start DNA Polymerase. ProtoScript II Reverse Transcriptase is a mutant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. OneTaq Hot Start DNA Polymerase is mixture of a Hot Start Taq DNA Polymerase combined with a proof-reading DNA polymerase, resulting in high-yield amplification with minimal optimization. The OneTaq One-Step RT-PCR Kit is capable of amplifying long transcripts up to 9 kb in length.

Two optimized reaction mixes are included, OneTaq One-Step Reaction Mix and Quick-Load OneTaq One-Step Reaction Mix. The reaction mixes offer robust conditions for both cDNA synthesis and PCR amplification. The unique OneTaq Quick-Load One-Step Reaction Mix contains additional dyes, offering color indication for reaction setup as well as direct gel loading.

Both total RNA and mRNA can be used as template. The kit can detect a GAPDH target as low as 0.1 pg per reaction. It can routinely detect RNA targets up to 9 kb. The OneTaq One-Step RT-PCR Kit is capable of multiplex detection of two or three targets.

Figure 1. Detection of RNA templates of different length.Figure 1. Detection of RNA templates of different length.

About 100 ng of Jurkat total RNA was used in 50 μl reactions following the standard protocol. The target sizes were Lane 1: 0.7 kb, Lane 2: 1.1 kb, Lane 3: 1.9 kb, Lane 4: 2.3 kb, Lane 5: 2.5 kb, Lane 6: 5.5 kb, Lane 7: 7.6 kb and Lane 8: 9.3 kb. The marker lane (M) contains Quick-Load 1 kb Plus DNA Ladder (NEB #N0469).
Figure 2. Sensitive detection of RNA down to 1 pg.Figure 2. Sensitive detection of RNA down to 1 pg.

Serial dilution of HeLa total RNA (Lane 1: 1 μg, Lane 2: 100 ng, Lane 3: 10 ng, Lane 4: 1 ng, Lane 5: 100 pg, Lane 6: 10 pg, Lane 7: 1 pg, Lane 8: 0.1 pg) was used in 50 μl reactions following the Standard Protocol. The marker lane (M) contains Quick-Load 1 kb Plus DNA Ladder (NEB #N0469).
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:
RT-PCR,
RT-PCR & cDNA Synthesis,
PCR

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E5315S     -20    
      OneTaq One-Step Enzyme Mix M1203AVIAL -20 1 x 0.06 ml 25 X
      OneTaq One-Step Reaction Mix M1204AVIAL -20 1 x 0.8 ml 2 X
      Quick-Load® OneTaq One-Step Reaction Mix M1205AVIAL -20 1 x 0.8 ml 2 X
      Nuclease-free Water B1502AVIAL -20 1 x 1.5 ml Not Applicable

Properties & Usage

Method Overview

N/A

References

  1. Liao, J. and Gong, Z. (1997). Biotechniques. 23, 368-370.
  2. Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual (3rd Ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
  3. Sellner, L N. and Turbett, G. R. (1998). Biotechniques. 25, 230-234.

Protocols, Manuals & Usage

Protocols

  1. Reaction Conditions (E5315)
  2. Standard PCR Protocol (E5315)
  3. One-Step RT-PCR Protocols (E5315)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

FAQs & Troubleshooting

FAQs

  1. How do I choose between One-Step RT-PCR and Two-Step RT-PCR protocols?
  2. What’s the difference between NEB #E6560 and NEB #E5315?
  3. How does One-Step RT-PCR kit protocol work for NEB #E5315?
  4. Can I use a 25 μl reaction volume rather than a 50 μl reaction volume using NEB #E5315?
  5. What conditions should I use for cDNA synthesis?
  6. Do I need to optimize the reaction components?
  7. How much RNA template should I use?
  8. How should I design my primers?
  9. What’s the recommended primer concentration?
  10. How many PCR cycles should I do?
  11. How do I choose between LunaScript Multiplex One-Step RT-PCR Kit and OneTaq One-Step RT-PCR Kit (NEB #E5315)?

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.

Licenses

Notice to Purchaser: Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the OneTaq® One-Step RT-PCR Kit for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com. 

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