New England Biolabs, M0439L, WarmStart® RTx Reverse Transcriptase (Glycerol-free)

Related Categories cDNA Synthesis & Reverse Transcriptases,, Isothermal Amplification & Strand Displacement,, PCR, qPCR & Amplification Technologies Applications Loop-Mediated Isothermal Amplification,, DNA Amplification, PCR & qPCR Specification Materials Required but not Supplied Deoxynucleotide (dNTP) Solution Mix (NEB #N0447) Nuclease-free Water (NEB #B1500) RNase Inhibitor, Murine (NEB #M0314) FAQ 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: 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: Does WarmStart® RTx have RNase H activity? A: Yes, WarmStart® RTx contains an active RNase H domain. 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: What are Hot Start and WarmStart® polymerases and when would I use them? A: When setting up room temperature reactions off ice When non-specific amplification is observed What does Hot Start/ Warm Start mean? Our Hot Start and WarmStart polymerases utilize aptamers that inhibit enzyme activity at room temperature, which discourages the formation of nonspecific products. The presence of these aptamers does not alter core enzyme function. The distinction between Hot Start and WarmStart is that Hot Start enzymes are thermophilic while WarmStart® enzymes are mesophilic; the thermodynamic range of the aptamers for Hot Start and WarmStart enzymes are largely similar. Aptamers are engineered oligonucleotides that bind to a specific target molecule through non-covalent interactions and include specific nucleobase modifications that can improve inhibition profiles and/or reduce unintended side effects. The benefit of aptamer-based inhibition over other Hot Start technologies (like antibodies) is that they do not require an activation step and bind reversibly in a temperature-dependent manner. NEB offers polymerases with aptamers for routine PCR, high-fidelity PCR, isothermal amplification, and reverse transcription. These aptamers can target different enzymatic functions for different polymerases. Learn More Why is it called "Hot Start?" Like many non-proofreading, Family A DNA polymerases, Taq Polymerase possesses the ability to add bases onto the end of ssDNA in a template-independent manner even at room temperature, and can result in the addition of non-specific bases onto the ends of DNA primers in the reaction, enabling off-target hybridization and reduced overall reaction specificity. In contrast, at higher temperatures, nonspecific binding is reduced as annealing becomes more stringent. Early methods to mitigate undesired activity at low temperature focused on the exclusion of key reaction components until the reaction temperature was increased, which could then be spiked into the mixture, triggering the reaction under a more restrictive, high temperature condition. Read our feature article, Using aptamers to control enzyme activities Hot Start Taq and beyond, to see data comparing product formation for enzymes with and without aptamer-based inhibition of activity. Q: What is the difference between buffers B1714: 10X Isothermal Amplification Buffer (Lyo-compatible) and B0537: 10X Isothermal Amplification Buffer? A: The glycerol-free enzyme will be active in both isothermal amplification buffers but the lyo-compatible buffer (NEB #B1714) is optimized for use in lyophilization workflows. Q: What is the Mg2+ concentration in the 10X Isothermal Amplification Buffer (Lyo-compatible)? A: The 10X Isothermal Amplification Buffer (Lyo-compatible) contains 80 mM MgSO4 and thus 8 mM MgSO4 in the 1X final reaction. Q: Does B1714: 10X Isothermal Amplification Buffer (Lyo-compatible) include any excipients? A: No, 10X Isothermal Amplification Buffer (Lyo-compatible) does not include any excipients for lyophilization. Excipients should be screened and selected by the end user in the application of interest. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-469750017 -1040178053 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman",serif; mso-fareast-font-family:"Times New Roman";}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-font-kerning:0pt; mso-ligatures:none;}div.WordSection1 {page:WordSection1; Q: Can WarmStart® RTx Reverse Transcriptase (Glycerol-free) at 75,000 U/mL be diluted prior to use? A: Yes, the 75,000 U/ml enzyme can be diluted in 1X Isothermal Amplification Buffer (Lyo-compatible) to 15,000 U/mL prior to use. The 15,000 U/ml dilution is stable at 4°C for at least 7 days. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-469750017 -1040178053 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman",serif; mso-fareast-font-family:"Times New Roman";}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-font-kerning:0pt; mso-ligatures:none;}div.WordSection1 {page:WordSection1;} Q: How many Freeze/Thaw cycles can WarmStart® RTx Reverse Transcriptase (Glycerol-free) tolerate? A: WarmStart® RTx Reverse Transcriptase (Glycerol-free) was tested up to 10 Freeze/Thaws at 75,000 U/ml and showed no activity loss in an RT-LAMP reaction. However, repeated freezing and thawing of this product should be avoided. The enzyme (75,000 U/ml) may be stored at 4°C for up to one month. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-469750017 -1040178053 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman",serif; mso-fareast-font-family:"Times New Roman";}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-font-kerning:0pt; mso-ligatures:none;}div.WordSection1 {page:WordSection1;}