The WarmStart Fluorescent LAMP/RT-LAMP Kit (with UDG) is designed to provide a simple, one-step solution for real-time detection of LAMP/RT-LAMP reactions via fluorescence. 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 kit is supplied with the WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) (NEB #M1708) featuring 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. Also included is a 50X LAMP Fluorescent Dye for real-time fluorescence measurement.
The inclusion of dUTP and thermolabile UDG in the master 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 also offered in a lyophilized format for ambient/room temperature storage and shipping and streamlined assay development. The lyophilized product has been developed to retain the performance of the E1708 liquid version and is offered in glass vials and a 96-well plate. To learn about the lyophilization process, or to inquire about customizing this product to meet your assay needs, please visit www.neb.com/lyoprime.
Figure 1: 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 2: 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 3: 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.
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Specifications & Change Notifications
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]
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]
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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®Fluorescent LAMP/RT-LAMP Kit (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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