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ProtoScript® II First Strand cDNA Synthesis Kit

Catalog # Concentration Size List Price Quantity Your Price
E6560L 150 reactions $1,103.00
$992.70
E6560S 30 reactions $275.00
$247.50
Catalog # Size List Price Your Price
E6560L 150 reactions $1,103.00
$992.70
E6560S 30 reactions $275.00
$247.50
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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
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    Avoiding RNase Contamination
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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  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
  Oligo d(T)23 VN S1332AVIAL -20 1 x 0.07 ml 50 µM
  ProtoScript® II Reaction Mix M0555AVIAL -20 5 x 0.4 ml 2 X
  ProtoScript® II Enzyme Mix M0556AVIAL -20 5 x 0.06 ml 10 X
  Random Primer Mix S1330AVIAL -20 5 x 0.07 ml 60 µM
  Nuclease-free Water B1502AVIAL -20 5 x 1.5 ml
  Oligo d(T)23 VN S1332AVIAL -20 5 x 0.07 ml 50 µM

Properties & Usage



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References
  • Liao, J. and Gong, Z. (1997). Biotechniques. 23, 368-370.
  • 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.
Quality Control Assay
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 Data Sheets
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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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