New England Biolabs, M0380L, WarmStart® RTx Reverse Transcriptase

This product is available in a Related Categories RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications Strand Displacement Amplification & Nicking Enzyme,, cDNA Synthesis,, Reverse Transcription (cDNA Synthesis), Specification 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 20 minutes at 50°C using poly(rA)•oligo(dT)18 as template. Reaction Conditions 1X Isothermal Amplification Buffer Pack 1X Isothermal Amplification Buffer Pack 20 mM Tris-HCl 10 mM (NH4)2SO4 50 mM KCl 2 mM MgSO4 0.1% Tween® 20 (pH 8.8 @ 25°C) Storage Buffer 100 mM KCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 10 minutes Unit Assay Conditions 20 mM Tris-HCl (pH 8.8), 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween-20, 0.5 mM dTTP, 0.4 mM poly(rA)•oligo(dT)18. FAQ Q: The product size was previously listed in Reactions and is now listed in Units. What has changed? A: To standardize product size and provide flexibility to usage, we have changed the product size to be listed in terms of Units. This will result in the following changes: M0380S: 50 Reactions changed to 400 Units M0380L: 250 Reactions changed to 2,000 Units In both instances, the volume of supplied product increased slightly to accommodate the change to Units. Q: What is LAMP and RT-LAMP? A: Loop Mediated Isothermal Amplification (LAMP) is an isothermal amplification method designed to detect a target nucleic acid without requiring sophisticated equipment. It uses a stand-displacing DNA polymerase such as a Bst DNA Polymerase and 4-6 primers recognizing 6-8 distinct regions of target DNA for a highly specific amplification reaction. LAMP provides high sensitivity (to fg or <10 copies of target) but with rapid results: reactions can be performed in as little as 5–10 minutes. Reactions can be performed with limited resources, using a water bath for incubation and detection of results by eye, or with real-time measurement and high-throughput instruments. Detection of RNA targets is accomplished by simple addition of a reverse transcriptase to the LAMP reaction, with RT-LAMP performed as a true one-step, isothermal workflow. WarmStart RTx Reverse Transcriptase (NEB #M0380) is a RNA-directed DNA polymerase coupled with a reversibly-bound aptamer that inhibits RTx activity below 40°C, making it particularly well suited for RT-LAMP. To learn more and to view our LAMP product offerings, please visit the LAMP Application Overview Page. Q: What advantages does WarmStart® RTx provide? A: Similar to use of hot-start DNA polymerases, a warm-start reverse transcriptase enables consistent performance in amplification reactions. With the WarmStart® property, any RT-LAMP reaction using either WarmStart RTx or Bst 2.0 WarmStart DNA Polymerase (NEB #M0538) can be set up at room temperature, without off-target amplification. This control provides a greater degree of specificity to RNA detection and amplification reactions, and enables flexibility in high-throughput or ice-free setup. Q: How sensitive is RNA amplification with WarmStart® RTx? A: One-step RT-LAMP reactions with WarmStart RTx can routinely provide target amplification with as little as 1 pg of input RNA. RT-PCR reactions with either WarmStart RTx or Hot Start Taq can provide target amplification with as little as 10 fg of input RNA. Q: Can WarmStart® RTx be inactivated? A: We recommend heating to 80°C for 10 minutes. Q: Is WarmStart® RTx active in other buffers? A: Yes, WarmStart® RTx is generally active in buffers with pH >7.5, 10–200 mM monovalent salt, and 2–20 mM Mg2+. Activity in other NEB Buffers: NEBuffer1.1: 10% NEBuffer2.1: 75% NEBuffer3.1: 75% CutSmart™ Buffer: 90% ThermoPol™: 90% Q: How do I use WarmStart® RTx in RT-LAMP? A: Prepare a 25 µL mix containing: 2.5 µL Isothermal Amplification Buffer (1X) 6 mM MgSO4 (8 mM total) 1.4 mM dNTP 1.6 µM FIP/BIP, 0.2 µM F3/B3, 0.4 µM LoopF/B 8 U Bst 2.0 WarmStart® or Bst 2.0® 7.5 U WarmStart® RTx Mix, place directly at 65°C Q: What is the maximum length of cDNA product produced by WarmStart® RTx? A: WarmStart RTx was developed for best performance with short amplicons in diagnostic amplification reactions (e.g. RT-LAMP and RT-PCR). However, it performs well in cDNA synthesis, with products up to 5 kb. For longer products, we recommend ProtoScript® II (NEB #M0368) or the convenient ProtoScript II First Strand cDNA Synthesis Kit (NEB #E6560). For the longest cDNA products, we would recommend Induro® Reverse Transcriptase (NEB #M0681), a group II intron-encoded RT. Q: How do I use WarmStart® RTx for cDNA synthesis? A: Prepare a 20 µL mix containing: 2 µL Isothermal Amplification Buffer (1X) 0.5 mM dNTP 6 µM Random Primer Mix 20 U Murine RNase Inhibitor 3.75 U WarmStart® RTx Mix, incubate 5 min at 25°C for annealing and 10 min at 55°C for synthesis. Inactivate RTx with 10 min at 80°C. Q: Does WarmStart® RTx have RNase H activity? A: Yes, WarmStart® RTx contains an active RNase H domain. Q: What dNTPs do you recommend for use with WarmStart® RTx? A: We provide dNTPs in two formats. They can be ordered as a convenient mix (Deoxynucleotide Solution Mix, (NEB #N0447), with a 10 mM concentration of each nucleotide) or as a set of 4 individual tubes (Deoxynucleotide Solution Set, (NEB #N0446), with a 100 mM concentration of each nucleotide). Q: Can DNA be used as a template for WarmStart® RTx? A: Yes, but the reaction is much less efficient compared to RNA-directed DNA synthesis. DNA-directed DNA synthesis is ~10-20% of the activity of RNA-directed at 45°C and ~50% at 55°C. Q: Does NEB have a master mix for LAMP or RT-LAMP reactions? A: Yes. We offer several options to support LAMP/RT-LAMP protocols. The WarmStart® LAMP Kit (DNA & RNA) (NEB #E1700) includes a 50X LAMP fluorescent dye to support fluorescent detection of LAMP/RT-LAMP reactions. The WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB #M1800) includes a pH-based colorimetric indicator for visual detection of LAMP/RT-LAMP reactions (a pink-to-yellow color change signifies amplification). Updated versions of both products include thermolabile UDG and dUTP to reduce the risk of carryover contamination (NEB #E1708 and NEB #M1804, respectively). In addition, the WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) (NEB #M1708) is compatible with different sample input types and supports multiple detection methods, including hydroxynaphthol blue. All LAMP master mixes include a combination of WarmStart RTx Reverse Transcriptase and Bst 2.0 WarmStart DNA Polymerase for fast and robust amplification from both DNA and RNA targets. Each mix requires only user-supplied LAMP primers and target DNA or RNA samples.