Product Class: Kit

WarmStart® Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG)

Need assistance designing LAMP primers? Use the NEB LAMP Primer Design Tool. 


Catalog #M1708

Product Introduction

  • Simple, one-step solution for Loop-Mediated Isothermal Amplification (LAMP) of DNA or RNA (RT-LAMP) targets
  • Reduce risk of carryover contamination with thermolabile UDG and dUTP included in the mix
  • Fully buffered master mix is compatible with different sample types and supports multiple detection methods
  • WarmStart technology inhibits activity at room temperature
  • Learn more about how RT-LAMP is being used for rapid COVID-19 screening
  • Also available: lyophilized LAMP/RT-LAMP mix
 

Product Information

Description

The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is designed to provide a simple, one-step solution for LAMP/RT-LAMP of DNA or RNA targets. LAMP and RT-LAMP are commonly used isothermal amplification techniques that provide rapid detection of a target nucleic acid using LAMP-specific primers (supplied by the user) and a strand-displacing DNA polymerase. This master mix features a blend of Bst 2.0 WarmStart® DNA Polymerase and WarmStart RTx Reverse Transcriptase in an optimized LAMP buffer solution. Both Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase have been engineered for improved performance in LAMP and RT-LAMP reactions. The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is fully buffered and compatible with different sample types, enabling multiple detection methods including turbidity detection, real-time fluorescence detection (when used with LAMP fluorescent dye) and end-point visualization such as colorimetric detection via a metal indicator (e.g., hydroxynaphthol blue.)

The inclusion of dUTP and thermolabile UDG in the mix reduces the possibility of carryover contamination, where unintended product of a previous amplification can serve as the substrate of a subsequent reaction. Thermolabile UDG is completely inactivated at temperatures above 50°C, thereby having no effect on the reaction.

This product is a core component of the LyoPrime WarmStart® Fluorescent LAMP/RT-LAMP Mix (with UDG), a lyophilized LAMP mix offering ambient/room temperature storage and shipping and streamlined assay development. To learn about the lyophilization process, please visit www.neb.com/lyoprime.

Figure 1: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with multiple detection methods



Amplification of target RNA or DNA by the WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) (NEB #M1708) can be confirmed using a variety of real-time (A) and endpoint (B) detection methods. Some of these detection modalities include fluorescence, non-pH-based colorimetric (e.g., hydroxynaphthol blue), turbidity or agarose gel electrophoresis. For real-time fluorescence detection, the master mix is available as a kit (NEB #E1708) that includes 50X LAMP Fluorescent Dye, which can be monitored in the SYBR®/FAM channel of most real-time PCR instruments.



Figure 2: LAMP/RT-LAMP master mixes and sample type considerations



NEB’s pH-based colorimetric LAMP master mixes with UDG (NEB #M1804) or without UDG (NEB# M1800) are weakly buffered to allow for visual detection of amplification using a pH-sensitive dye. However, the low buffering capacity required to generate the pink to yellow color change limits sample compatibility with the pH-based colorimetric mixes, as highly buffered sample inputs or acidic samples may impact the color change. The multi-purpose LAMP/RT-LAMP 2X Master Mix with UDG (NEB #M1708, NEB #E1708) or without UDG (NEB #E1700) is fully buffered and can more readily tolerate these types of sample inputs, making it compatible with various detection modes including fluorescence or other colorimetric dyes (e.g., hydroxynaphthol blue).



Figure 3: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) provides robust detection of human DNA and RNA targets



RT-LAMP (RNA targets) or LAMP (DNA targets) experiments were performed with NEB #M1700: WarmStart LAMP 2X Master Mix, which is the master mix in the NEB #E1700 kit that does not contain dUTP/UDG, and NEB #M1708: WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG). Reactions containing 1X LAMP primers and 1X LAMP Fluorescent dye were set up in quadruplicate over three logs of total Jurkat RNA or Jurkat DNA (10 ng to 0.1 ng) in 96-well, 25 µl reactions. Control reactions without template (NTC) were also evaluated.  Reactions were incubated at 65°C for 40 minutes and fluorescence was monitored every 15 seconds in the SYBR/FAM channel of a real-time thermocycler (Bio-Rad® CFX96). Each dot represents the time at which the fluorescence signal for a single reaction crosses the instrument-defined threshold.  All four replicates were detected at each template input unless otherwise indicated (note that dots frequently overlap given similar detection time for the replicates). Overall, similar performance was observed for both NEB #M1700 and NEB #M1708 at each template input.  No amplification was observed in any of the no template control reactions.



Figure 4: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with automated reaction assembly



LAMP assays targeting synthetic SARS-CoV-2 RNA were assembled either manually or with the TEMPEST® liquid handling platform (96-well, 25 µl reactions). The assay was carried out using either positive samples (human total RNA plus synthetic SARS-CoV-2 RNA at 5,000, 500 or 50 copies per reaction) or no template (NTCs) as indicated. Reactions were incubated at 65°C for 40 minutes and monitored with 1X LAMP Fluorescent dye in the SYBR/FAM channel of a real-time instrument (Bio-Rad CFX96). Similar time to detection and LOD results were observed for both reaction assembly methods.



Figure 5: The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) is compatible with non-pH-based colorimetric detection (e.g., hydroxynaphthol blue)


The WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) was used to amplify synthetic SARS-CoV-2 RNA using a dual primer mix that targets the N and E genes in the presence of 0.12 mM of hydroxynaphthol blue, a colorimetric metal indicator. The assay was carried out using either positive samples (human total RNA plus synthetic SARS-CoV-2 RNA at 5,000, 500 or 50 copies per reaction), negative samples (human total RNA alone) or no template (NTCs), as indicated. The 25 µl reactions were incubated at 65°C for 45 minutes and then visually inspected. 

A. All positive samples gave a positive result with robust color change from purple to blue, including detection in all samples at a low input of 50 copies per reaction (n = 20).

B. No color change was observed for numerous replicates of negative samples (n = 32).

This product is related to the following categories:
Isothermal Amplification & Strand Displacement Products,
Master Mixes Products,
This product can be used in the following applications:
Loop-Mediated Isothermal Amplification

Properties & Usage

Materials Required but not Supplied

LAMP Primers (we recommend using the NEB LAMP Primer Design Tool)
Target nucleic acid samples
Molecular Biology Grade H2O
Heat block, water bath, real-time turbidimeter or thermocycler (with real-time fluorescence measurement if desired) and instrument-appropriate reaction vessels
 

Protocols, Manuals & Usage

Protocols

  1. WarmStart® Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) Protocol (NEB #M1708)

Application Notes

FAQs & Troubleshooting

FAQs

  1. What is the optimal LAMP/RT-LAMP amplification temperature?
  2. What is the Mg++ concentration in the WarmStart LAMP/RT-LAMP Master Mix?
  3. What types of input samples/materials are compatible with the LAMP/RT-LAMP mixes?
  4. How fast should I expect a result?
  5. Can I set up my LAMP/RT-LAMP reactions at room temperature?
  6. The WarmStart LAMP Master Mix has some precipitation in the tube after thawing, is this normal?
  7. How do I design LAMP Primers?
  8. Does the WarmStart LAMP/RT-LAMP 2X Master Mix (with UDG) enable carryover prevention/contamination reduction?
  9. Amplification occurred in my NTC sample(s) following isothermal incubation. What happened?
  10. What detection methods can be used for LAMP/RT-LAMP experiments?
  11. What are the advantages of using standard LAMP (NEB #E1700, M1708, E1708) over pH-based colorimetric LAMP (NEB #M1800, M1804)?
  12. Is a separate incubation step required for RT-LAMP?
  13. What type of purification is recommended for LAMP primers?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased product to perform loop-mediated amplification (“LAMP”) and reverse transcription loop-mediated amplification (“RT-LAMP”) for research use only. LAMP is a patented technology belonging to Eiken Chemical Co., Ltd and any other use other than research may require a license from Eiken Chemical Co., Ltd. 

Nucleic acid-based aptamers for use with thermophilic DNA polymerases and reverse transcriptases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the WarmStart® Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) for Research Use Only (RUO).  Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com.

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