New England Biolabs, M1800L, WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA)

Simple, one-step solution for Loop-Mediated Isothermal Amplification (LAMP) of DNA or RNA (RT-LAMP) targets Related Categories Isothermal Amplification & Strand Displacement Applications Loop-Mediated Isothermal Amplification,, Isothermal Amplification FAQ Q: What is the difference between using Colorimetric LAMP and regular LAMP reactions? A: Our pH-based colorimetric LAMP technology is based on the same principle of LAMP and RT-LAMP amplification, but has been designed to allow visual detection of positive reactions. The WarmStart Colorimetric LAMP 2X Master Mixes contains the pH-sensitive dye Phenol Red in a low-buffer reaction solution that changes color from bright pink to yellow upon DNA amplification (due to the release of a proton per nucleotide incorporated). This detection makes it possible to use simple equipment (e.g., hot water for incubation, visual detection) instead of sophisticated and expensive instruments for LAMP reactions. Q: How long should I incubate the Colorimetric LAMP reaction before checking the results? A: Our recommended incubation time for colorimetric LAMP is 30 min. At this end point, most positive LAMP reactions will have changed color and non-template controls will be negative. If desired, the time can be shortened to 15–20 minutes with good assays or high copy targets, or lengthened to up to 60 minutes with very low input, impure samples, and slower assays. If optimizing reaction time, progress can be simply checked by looking at the reaction colors and incubating longer if incomplete. Note that when extending the reaction time, NTCs must also be monitored to ensure that false positives are not present. Q: What components are contained in the WarmStart® Colorimetric LAMP 2X Master Mix and what else do I need to perform a LAMP reaction? A: The WarmStart Colorimetric 2X Master Mix contains a low-Tris reaction buffer with all necessary cofactors at optimized 2X concentrations for LAMP including Bst 2.0 WarmStart® DNA Polymerase, WarmStart RTx and Phenol Red for pH detection of LAMP. To perform a LAMP or RT-LAMP reaction, add primers, target DNA or RNA and adjust total reaction volume to a final concentration of 1X. Detailed instructions can be found in the protocol. Q: What is the Mg++ concentration in the Colorimetric LAMP 2X Master Mix? A: The 2X Mix contains 16.0 mM MgSO4 and thus 8.0 mM MgSO4 in the 1X final reaction. Q: Can the Colorimetric LAMP 2X Master Mix be used in instruments that utilize fluorescent detection of DNA amplification or real time turbidity measurement? A: The master mix is compatible with fluorescent detection methods when supplemented with DNA intercalating fluorescent dyes and it gives reaction speed similar to conventional LAMP. It is also possible to perform LAMP detection on a real time turbidimeter, although its reaction speed is slightly slower than that of conventional LAMP. Q: How do I design LAMP Primers? A: LAMP primers can be challenging to design manually, and software programs are strongly recommended for both ease of design and likelihood of reaction success. We recommend using the NEB LAMP Primer Design Tool. As performance and levels of non-template amplification can vary even with in silico design, we recommend evaluating 2–4 complete sets of LAMP primers for optimal sensitivity and specificity before choosing a final set. For an overview of how LAMP primers are designed and utilized, please watch our LAMP Primer Design Tool Tutorial, and read this application note for more details. Q: The WarmStart LAMP Master Mix has some precipitation in the tube after thawing, is this normal? A: Yes, precipitation of the LAMP Master Mixes after freezing/thawing is normal. It is important to resuspend the mix by thoroughly mixing or vortexing to dissolve all the precipitate material, but after suspension the Master Mixes can be used as normal. Q: How stable are the WarmStart Colorimetric LAMP 2X Master Mixes? A: The Colorimetric LAMP Mixes are stable under standard handling and usage conditions. They contain a low concentration of Tris pH 8 and the pH-sensitive Phenol Red shows bright pink color near this pH. After repeated usage of the same tube, a slight drop of pH is possible but will typically not impact reaction performance provided that the mix is still pink prior to amplification. Occasionally, after multiple uses and extended exposure to air, the pH may drop to below pH 7, and the mix will turn a light orange to yellow color before use. This color change provides a visual indication that the solution is no longer suitable for the colorimetric LAMP reaction. Thus it is recommended to avoid extended exposure to air by closing tube caps and storing the master mix in the vial it was provided in (or in similar vials with O-rings) at -20 °C. If the master mix is pipetted into strip tubes, PCR tubes or similar, we recommend using the material within the same day. Q: What buffer should I use for my target samples and primers going into the colorimetric LAMP reaction? A: We recommend dissolving or diluting target DNA or RNA in H2O and adding 1–5 µl to 25 µl LAMP reactions. TE buffer (10 mM Tris, 1.0 mM EDTA, pH 7.5–8.0) can be used, but targets dissolved in TE should not be added in volumes higher than 1–2 µl (or <10% of final volume). Similarly, any sample stored in alkaline (pH greater than 8.5) or acidic (pH less than 7) solution should make up no more than 10% of the final reaction volume as a higher amount may affect the pH-based detection. If >5 μl is needed for sufficient copy number, use water or a 0.1X solution of sample buffer. Q: What components are contained in the WarmStart® Colorimetric LAMP Master Mix and what else I need to perform a LAMP reaction? A: The WarmStart Colorimetric Master Mix contains a low-Tris reaction buffer with all necessary cofactors at optimized 2X concentrations for LAMP, Bst 2.0 WarmStart® DNA Polymerase, WarmStart RTx and Phenol Red for pH detection of LAMP. To perform a LAMP or RT-LAMP reaction, the user adds primers, target DNA or RNA and adjusts to a final concentration of 1X. Detailed instructions can be found in the protocol. Q: What is the difference between the WarmStart® Colorimetric LAMP 2X Master Mix with UDG (NEB #M1804) and the WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB #M1800)? A: Both WarmStart Colorimetric LAMP 2X Master Mixes contain a pH-sensitive dye in a low-buffer reaction solution that changes color from bright pink to yellow upon DNA amplification. This simple visual detection combined with a single temperature incubation makes it possible to use less sophisticated equipment (heat blocks, incubators, etc) than with typical LAMP reactions. The WarmStart® Colorimetric LAMP 2X Master Mix with UDG is a modified version of M1800 that contains dUTP and Antarctic Thermolabile UDG (NEB #M0372), which prevents amplification of previous reaction products that contain uracil. These additions reduce the possibility of carryover contamination (where the product of a previous reaction can unexpectedly serve as the substrate of a subsequent reaction). Both mixes contain WarmStart Bst 2.0® and WarmStart RTx and can be used to amplify both RNA and DNA/cDNA targets. Q: What if my colorimetric LAMP mix is orange instead of pink prior to amplification? A: The Colorimetric LAMP Mixes are stable under standard handling and usage conditions. They contain a low concentration of Tris pH 8 and the pH-sensitive Phenol Red shows bright pink color near this pH. After repeated usage of the same tube, a slight drop of pH is possible but will typically not impact reaction performance provided that the mix is still pink prior to amplification. Occasionally, after multiple uses and extended exposure to air, the pH may drop to below pH 7, and the mix will turn a light orange to yellow color before use. This color change provides a visual indication that the solution is no longer suitable for the colorimetric LAMP reaction. Thus it is recommended to avoid extended exposure to air by closing tube caps and storing the master mix in the vial it was provided in (or in similar vials with O-rings) at -20 °C. If the master mix is pipetted into strip tubes, PCR tubes or similar, we recommend using the material within the same day. Q: Should I use Guanidine HCl (B2619A) in colorimetric LAMP reactions? A: We recommend colorimetric LAMP reactions contain a final concentration of 40 mM Guanidine HCl and accordingly provide it for addition to the LAMP reaction in the standard SARS-CoV-2 workflow. However, some upstream sample prep workflows may introduce a source of guanidine into the sample which can be carried into the LAMP reaction. If guanidine will be carried into the reaction with the input material, we recommend not exceeding 60 mM in the final reaction and replacing the volume allotted for guanidine HCl with nuclease-free water. Guanidine enhances isothermal amplification, yielding faster detection of SARS-CoV-2 RNA. Colorimetric LAMP was carried out using purified positive samples (10 ng/ul human total RNA + synthetic SARS-CoV-2 RNA at 10,000 copies per reaction). E1 or N2 primer sets from the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit were used as indicated, and amplification was monitored in real time using a fluorescent dye. For both primer sets, addition of guanidine HCl to a final concentration of 40-60 mM enhanced amplification, resulting in earlier detection and enhanced color change. Q: How much sample can be added to the colorimetric LAMP reaction as input? A: For best results, we recommend 2 µl of extracted nucleic acid in a 25 µl reaction. Larger or smaller volumes of samples may be tolerated. Ensure the nucleic acid is eluted or prepared in water to avoid carrying over excess buffer to the reaction. Material eluted in TE or similar elution buffer should be kept to less than 5 µl (20% v/v) of the final reaction volume. Samples in transport media (VTM or UTM) should be kept to less than 2 µl (8% v/v). To increase sample volume, use 0.1X Elution Buffer or water for preparation of the nucleic acid. Q: Can I monitor the color change of the colorimetric LAMP reactions using a spectrophotometer? A: Yes. The reaction can be monitored in real time or at end point as part of a high-throughput workflow by measuring the absorbance ratio of 432 nm/560 nm. Q: Can the WarmStart Colorimetric LAMP 2X Master Mixes be used in instruments that utilize fluorescence detection? A: The colorimetric master mix is compatible with fluorescent detection methods when supplemented with a DNA intercalating fluorescent dyes (SYTO 9 or similar) and it gives reaction speeds similar to conventional LAMP. However, we recommend using our standard LAMP products (NEB #E1700, E1708) when the primary readout is fluorescence. Q: What is the optimal LAMP/RT-LAMP amplification temperature? A: We recommend 65°C as the initial LAMP temperature. However, it is possible to perform LAMP and RT-LAMP reactions from 55–70 °C, depending on the nature of the primer set and targets. Q: How fast should I expect a result? A: Target amplification will vary depending on a number of factors including primer design (We recommend using the NEB LAMP Primer Design Tool.), template purity and template quantity. We suggest beginning with an incubation time of 30 minutes. If desired, the time can be shortened to 15–20 minutes with optimized assays or high copy targets. Alternatively, it can be lengthened to up to 60 minutes for low inputs, impure samples, and/or slower assays. Note that when extending the reaction time, NTCs must also be monitored to ensure that false positives are not an issue. Q: Can I set up my LAMP/RT-LAMP reactions at room temperature? A: Yes – the Bst DNA polymerase and reverse transcriptase enzymes used in this master mix are WarmStart® formulations, so they are inhibited by modified aptamers at room temperature. Accordingly, reactions can be prepared at room temperature without significantly affecting LAMP activity. For additional information about modulating enzyme activity at room temperature with aptamers, please see “Using aptamers to control enzyme activities: Hot Start Taq and beyond”. Q: Amplification occurred in my NTC sample(s) following isothermal incubation. What happened? A: Amplification in the non-template control within 30 minutes may indicate cross-contamination during reaction set up or a systemic issue. Some primer sets are more susceptible to non-specific amplification than others. We recommend evaluating at least two LAMP primer sets for any given target. In addition, some reaction formats or workflows may be more prone to non-specific amplification with particular primer sets such as: Large reaction volumes in small vessels (e.g., 20 uL in 384-well plates) Low reaction temperatures (e.g., 60°C instead of 65°C) If using a real-time thermocycler, a melt or denaturation curve can be included after the LAMP incubation to distinguish between spurious amplification and cross-contamination since each LAMP amplicon will produce a unique melt profile. If the NTC reaction is positive upon repeat testing and/or workflow changes, replace all reagent stocks and clean workspace with an acceptable surface decontaminate such as 10% bleach (1:10 dilution of commercial 5.25-6.0% sodium hypochlorite). Q: Is a separate incubation step required for RT-LAMP? A: A separate reverse transcription step is not necessary to perform RT-LAMP as WarmStart RTx Reverse Transcriptase will produce cDNA during isothermal incubation at 65°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. 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 type of purification is recommended for LAMP primers? A: When screening multiple sets of LAMP primers to identify ones with optimal performance for a given target, standard desalting is generally sufficient. However, we would recommend PAGE or HPLC purification of the FIP and BIP primers at a minimum for the final assay to ensure robustness with respect to time to detection and sensitivity.