Description:

ProtoScript® II Reverse Transcriptaseis a recombinant M-MuLV reverse transcriptasewith reduced RNase H activity and increasedthermostability. It can be used to synthesize firststrand cDNA at higher temperatures than the wildtype M-MuLV. The enzyme is active up to 48°C,providing higher specificity, higher yield of cDNAand more full-length cDNA product up to 12 kb.Protoscript II Reverse Transcriptase performs as well as other RNase H^– Reverse TranscriptasesJurkat 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 templatesJurkat 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 RNADecreasing 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 sensitivityDecreasing 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.

Reagents Supplied

The following reagents are supplied with this product:

	Store at (°C)	Concentration
ProtoScript® II Reverse Transcriptase Reaction Buffer	-20	5X
DTT (100mM)	-20	10X

Notes:

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.