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
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
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
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
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
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Quality Control Assays
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Specifications & Change Notifications
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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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