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Reverse Transcription / cDNA Synthesis

ProtoScript® II Reverse Transcriptase

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.

Advantages:

  • High yield cDNA synthesis up to 12 kb
  • Increased thermostability (up to 48°C !)
  • Excellent sensitivity (Input Total RNA < 1pg)
  • Suitable for RNA that contains high amounts of secondary structure

Performance

Protoscript II Reverse Transcriptase performs as well as other RNase H– Reverse Transcriptases

NEB_MuLVRNaseH_image1a

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 for 35–40 cycles. Ladder L is the 2 Log DNA Ladder.

Robust cDNA synthesis is achieved even with longer templates

NEB_MuLVRNaseH_image2

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 for 35–40 cycles. Sizes are indicated above gel.

Generate high quality cDNA even with very low amounts of starting RNA

NEB_MuLVRNaseH_image3

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 for 40 cycles. The target is a 0.6 kb fragment of GAPDH. Ladder L is the 2-Log DNA Ladder.

ProtoScript II Reverse Transcriptase displays superior sensitivity

NEB_MuLVRNaseH_image4

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.

Available products

PRODUCT REF. SIZE PRICE MORE INFOS
ProtoScript® II Reverse Transcriptase M0368S 4 000 u In den Shop wechseln Produktinformationen
M0368L 10 000 u In den Shop wechseln Produktinformationen
M0368X 40 000 u In den Shop wechseln Produktinformationen
ProtoScript® II First Strand cDNA Synthesis Kit E6560S 30 reactions In den Shop wechseln Produktinformationen
E6560L 150 reactions In den Shop wechseln Produktinformationen
AMV Reverse Transcriptase (active from 60°C) M0277S 200 u In den Shop wechseln Produktinformationen
M0277L 1 000 u In den Shop wechseln Produktinformationen
M-MuLV Reverse Transcriptase (Rnase H+) M0253S 10 000 u In den Shop wechseln Produktinformationen
M0253L 50 000 u In den Shop wechseln Produktinformationen

Further information can be found in our Technical Resources section or at neb.com