Product Class: Kit

ProtoScript® II First Strand cDNA Synthesis Kit

Product Introduction

The ProtoScript® II First Strand cDNA Synthesis Kit is optimized mix for higher specificity and yield of cDNA.

  • Recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability (active up to 48°C)
  • Generates cDNA up to 10 kb
  • Supplied with Oligo-dT primers, Randomized Primer Mix, and nuclease-free water
Catalog # Size Concentration
E6560S 30.0 reactions
E6560L 150.0 reactions

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Product Information

Description

ProtoScript II First Strand cDNA Synthesis Kit features two optimized mixes, ProtoScript II Enzyme Mix and ProtoScript II Reaction Mix. The enzyme mix combines ProtoScript II Reverse Transcriptase and Murine RNase Inhibitor, while the reaction mix contains dNTPs and an optimized buffer. 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 and higher yield of cDNA.

The kit also provides two optimized primers for reverse transcription and nuclease-free water. An anchored Oligo-dT primer [d(T)23VN] forces the primer to anneal to the beginning of the polyA tail. The optimized Random Primer Mix provides random and consistent priming sites covering the entire RNA templates including both mRNAs and non-polyadenylated RNAs. The first strand cDNA product generated is up to 10 kb.

Figure 1: cDNA Synthesis of Jurkat RNA with the ProtoScript II First Strand cDNA Synthesis KitFigure 1: cDNA Synthesis of Jurkat RNA with the ProtoScript II First Strand cDNA Synthesis Kit

First strand cDNA synthesis was carried out in the presence of 1X ProtoScript II Reaction Mix and 1X
ProtoScript II Enzyme Mix at 42°C using 250 ng of Jurkat total RNA. Four amplicons from different
genes were amplified using 1X LongAmp® Taq 2X Master Mix (NEB #M0287). Lane 1, a 1.9 kb
amplicon from SDHA gene; Lane 2, a 5.5 kb from the guanine nucleotide exchange factor; Lane 3,
a 7.3 kb amplicon from Xrn-1 gene; and Lane 4, a 9.2 kb amplicon from fibrillin gene. Marker M is
the 1 kb Plus DNA Ladder (NEB #N3200).
Quantitative detection of reverse transcription products using the ProtoScript II First Strand cDNA Synthesis KitQuantitative detection of reverse transcription products using the ProtoScript II First Strand cDNA

Ten-fold serial dilutions of in vitro transcribed luciferase RNA (1x102 to 1x109 copies) were mixed with 1 ng Jurkat total RNA. The RNA was reverse transcribed using first strand cDNA synthesis kits, as indicated, and 1/10th of the cDNA product was used for qPCR using luciferase-specific primers and iTaqTM Universal SYBR Green Supermix (BioRad). Ct values were plotted as a function of input luciferase RNA equivalent copies. Each data point represents the mean +/- 95% confidence interval of a minimum of 5 amplification reactions. ProtoScript II – NEB ProtoScript II First Strand cDNA Synthesis Kit  and random primer mix; SuperScript II – Invitrogen SuperScript First Strand Synthesis System for RT-PCR and random hexamers; SuperScript® III – Invitrogen SuperScript III First Strand Synthesis SuperMix and random hexamers.
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:
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis),
RNA Cloning,
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
  • E6560S     -20    
      ProtoScript® II Reaction Mix M0555AVIAL -20 1 x 0.4 ml 2 X
      ProtoScript® II Enzyme Mix M0556AVIAL -20 1 x 0.06 ml 10 X
      Random Primer Mix S1330AVIAL -20 1 x 0.07 ml 60 µM
      Nuclease-free Water B1502AVIAL -20 1 x 1.5 ml Not Applicable
      Oligo d(T)23 VN S1332AVIAL -20 1 x 0.07 ml 50 µM
  • E6560L     -20    
      ProtoScript® II First Strand cDNA Synthesis Kit E6560S -20 5 x 30 reactions Not Applicable
      ProtoScript® II Reaction Mix M0555AVIAL -20 1 x 0.4 ml 2 X
      ProtoScript® II Enzyme Mix M0556AVIAL -20 1 x 0.06 ml 10 X
      Random Primer Mix S1330AVIAL -20 1 x 0.07 ml 60 µM
      Nuclease-free Water B1502AVIAL -20 1 x 1.5 ml Not Applicable
      Oligo d(T)23 VN S1332AVIAL -20 1 x 0.07 ml 50 µM

Properties & Usage

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.),. (pp. 8.46–8.53 avnd 11.37–11.42). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

Protocols, Manuals & Usage

Protocols

  1. First Strand cDNA Synthesis Protocols (E6560)

Manuals

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

Usage & Guidelines

FAQs & Troubleshooting

FAQs

  1. How do I choose between LunaScript® RT SuperMix Kit and ProtoScript first strand cDNA synthesis kits?
  2. What is the difference between E6560 and E6300?
  3. What is the reaction temperature for E6560?
  4. Is RNaseH treatment required before PCR amplification?
  5. Can the cDNA products be used in real-time PCR analysis?
  6. What thermostable DNA polymerase can be used for PCR after cDNA synthesis?
  7. How do I choose between Induro Reverse Transcriptase and the different LunaScript and ProtoScript products?
  8. Why do I have low cDNA yields?
  9. How do I know whether my template RNA is of good quality?

Troubleshooting

Problem Suggestion
Low Yield of cDNA Check the integrity of the RNA by denaturing agarose gel electrophoresis (2). RNA should have a minimum A260/A280 ratio of 1.7 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides (2).
  Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (2).
  Some target RNA may contain strong pauses for RT; Use random priming instead of d(T)23VN.
  Use sufficient amount of RNA.

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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