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.
Figure 1: 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).Figure 2: 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 LongAmpTaq Master Mix (NEB #M0533) for 35–40 cycles. Sizes are indicated above gel. Figure 3: 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).Figure 4: 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:
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
DTT
B1034AVIAL
-20
4 x 0.5 ml
0.1 M
ProtoScript® II Reverse Transcriptase
M0368LVIAL
-20
4 x 0.05 ml
200,000 units/ml
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.
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
Roth, M.J., Tanese, N. and Goff, S.P. (1985). J. Biol. Chem.. 260, 9326-9335.
Kotewicz, M.L. et al, (1988). Nuc. Acids Res.. 16, 265-277.
Lim, D. et al, (2006). J. Virol.. 80, 8379-8389.
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.
Terry Fei Fan Ng, Nikola O Kondov, Xutao Deng, Alison Van Eenennaam, Holly L Neibergs, Eric Delwart (2015) A metagenomics and case-control study to identify viruses associated with bovine respiratory disease. J Virol; 89, 5340-9. PubMedID: 25740998, DOI: 10.1128/JVI.00064-15
Quality, Safety & Legal
Quality Control Assays
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 & Change Notifications
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]
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]
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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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