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Reverse Transcriptases

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Avian Myeloblastosis Virus (AMV) Reverse Transcriptase (NEB #M0277) and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase (NEB #M0253) are RNA-directed DNA polymerases. These enzymes can synthesize a complementary DNA strand initiating from a primer using RNA (cDNA synthesis) or single-stranded DNA as a template. M-MuLV Reverse Transcriptase lacks 3´ → 5´ exonuclease activity. ProtoScript II Reverse Transcriptase (NEB  #M0368) 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 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.

Take advantage of the low cost per unit with ProtoScript II Reverse Transcriptase*

* Based on US list prices, as of 5/12.

Advantages of ProtoScript II Reverse Transcriptase:

  • High cDNA yield
  • Superior performance for longer templates
  • Increased thermostability
  • Value pricing

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 (NEB #M0368) (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® Hot Start Taq 2X Master Mix (NEB #M0533) for 35–40 cycles. Ladder L is the Quick-Load® 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 (NEB #M0368). 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. Ladder L is the 2-Log DNA Ladder (NEB #N0469).

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 (NEB #M0368) 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.

LongAmp® and Quick-Load® are registered trademarks of New England Biolabs, Inc.
SsoAdvanced™ is a trademark of BioRad, Inc.
SYBR® is a registered trademark of Life Technologies, Inc.

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