New England Biolabs, M0253S, M-MuLV Reverse Transcriptase

M-MuLV Reverse Transcriptase synthesizes a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template. Related Categories RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications cDNA Synthesis,, Reverse Transcription (cDNA Synthesis),, RT-PCR & cDNA Synthesis, Specification Materials Required but not Supplied RNase Inhibitor, Murine (NEB #M0314) Nuclease-free Water (NEB #B1500) 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) as template primer with 50 mM Tris-HCI (pH 8.3), 6 mM MgCl 2 , 10 mM dithiothreitol, 0.5 mM [ 3 H]-dTTP and 0.4 mM poly(rA) - oligo(dT)12-18. Reaction Conditions 1X M-MuLV Reverse Transcriptase Reaction Buffer Incubate at 42°C 1X M-MuLV Reverse Transcriptase Reaction Buffer 50 mM Tris-HCl 75 mM KCl 3 mM MgCl2 10 mM DTT (pH 8.3 @ 25°C) Storage Buffer 50 mM Tris-HCl 150 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% NP-40 pH 7.6 @ 25°C Heat Inactivation 65°C for 20 minutes Molecular Weight Theoretical: 78000 daltons FAQ Q: Can M-MuLV Reverse Transcriptase be used in other NEBuffers? A: Optimal activity is seen in its unique buffer. NEBuffer 2 gives around 50% activity. Optimal activity is seen at a Mg2+ concentration of 3 mM. At 10 mM Mg2+, activity drops to 60%. The salt optimum is tight around 75 mM (1). (1) Gerard G. F., and D' Alessio J. M., Chapter 6 (73-93) From: Methods in Molecular Biology, Vol.16: Enzymes of Molecular Biology Edited by: M. M. Burell 1993 Humana Press Inc. Totowa, NJ Q: Does M-MuLV Reverse Transcriptase require DTT? A: Yes (1). (1) Gerard G. F., and D' Alessio J. M., Chapter 6 (73-93) From: Methods in Molecular Biology, Vol.16: Enzymes of Molecular Biology Edited by: M. M. Burell 1993 Humana Press Inc. Totowa, NJ Q: Can the M-MuLV Reverse Transcriptase transcribe through RNA-DNA junctions? A: Yes. M-MuLV reverse transcriptase is able to transcribe across an RNA - DNA junction. Q: How can the length of the product generated by M-MuLV Reverse Transcriptase be increased? A: Incubation at 42°C can relax some RNA secondary structure allowing M-MuLV Reverse Transcriptase to produce longer products. Q: How can the yield be improved when using M-MuLV Reverse Transcriptase? A: Avoiding ribonuclease contamination is of primary concern, since high quality template RNA is required. The ProtoScript First Strand cDNA Synthesis Kit (NEB# E6500) includes an RNase inhibitor. Q: What are some of the reasons for M-MuLV Reverse Transcriptase reaction failure? A: SDS, DMSO, high salt, or EDTA in the nucleic acid template preparation can inhibit the reaction. M-MuLV Reverse Transcriptase is especially sensitive to the chelation effects of EDTA, since the Mg2+ concentration of 3 mM is optimal. Other molecular biology techniques typically use 10 mM Mg2+ so they are more able to tolerate traces of EDTA. Q: Is M-MuLV Reverse Transcriptase active at temperatures higher than 42°C? A: For specific difficult templates (e.g. with high GC content or significant secondary structure) a higher reaction temperature may be used but the overall yield of the DNA may be decreased. We have successfully copied 6 kb messages by reverse transcription at 55°C. Q: Can DNA be used as a template for M-MuLV Reverse Transcriptase? A: Yes, but the reaction is less efficient (1). (1) Gerard G. F., and D' Alessio J. M., Chapter 6 (73-93) From: Methods in Molecular Biology, Vol.16: Enzymes of Molecular Biology Edited by: M. M. Burell 1993 Humana Press Inc. Totowa, NJ Q: Are NEB DNA Polymerases supplied with dNTPs? A: No, the dNTPs must be ordered separately. They can be ordered as a convenient mix (Deoxynucleotide (dNTP) Solution Mix (NEB# N0447) with a 10 mM concentration of each deoxynucleotide) or as a set of 4 individual tubes (Deoxynucleotide (dNTP) Solution Set (NEB# N0446) with a 100mM concentration of each deoxynucleotide).