New England Biolabs, E1708L, WarmStart® Fluorescent LAMP/RT-LAMP Kit (with UDG)\n 

Related Categories Isothermal Amplification & Strand Displacement Applications Loop-Mediated Isothermal Amplification Specification Materials Required but not Supplied LAMP Primers (we recommend using the NEB LAMP Primer Design Tool ) Target nucleic acid samples Molecular Biology Grade H 2 O Heat block, water bath, real-time turbidimeter or thermocycler (with real-time fluorescence measurement if desired) and instrument-appropriate reaction vessels FAQ 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: What is the Mg++ concentration in the WarmStart LAMP/RT-LAMP Master Mix? A: The 2X Master Mix contains 16 mM MgSO4 and thus 8 mM MgSO4 in the 1X final reaction. Q: How do I use the fluorescent dye? A: The fluorescent dye can be detected using the SYBR®/FAM channel of common real-time PCR instruments. The intercalating fluorescent dye is provided at a 50X concentration and we suggest using 0.5 μl of dye per 25 μl LAMP reaction for monitoring amplification on most real-time qPCR instruments. In some situations, further dilution of the dye may be necessary (to a final concentration of 0.1-0.5X). Q: What types of input samples/materials are compatible with the LAMP/RT-LAMP mixes? A: LAMP is typically a robust and inhibitor tolerant technique and the WarmStart® LAMP/RT-LAMP Master Mix is capable of direct amplification using a variety of sample types. For highest specificity and sensitivity, we recommend purified nucleic acid as input. Most routine methods of template purification are sufficient, such as the Monarch® Nucleic Acid Extraction Kits. We suggest using 2 µl of extracted nucleic acid for a 25 µl LAMP reaction. Larger or smaller volumes of samples may be tolerated. Samples in transport media (VTM or UTM) should be kept to less than 2 µl (8% v/v). 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: How do I confirm a positive result? A: LAMP reactions produce a series of concatemers containing the target sequence that vary in size. Because the LAMP amplicons contain repeats of the same DNA sequence, they typically have a unique melting temperature that can confirm amplification of the correct target. If using real-time fluorescence, a melt or denaturation curve can be included after the LAMP incubation. For more information, see the Troubleshooting section in the WarmStart LAMP Kit Manual. 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: 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 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: What is the difference between NEB #E1708: WarmStart Fluorescent LAMP/RT-LAMP Kit (with UDG) and NEB #E1700: WarmStart LAMP Kit (DNA & RNA)? A: Both LAMP Kits include a 2X master mix containing Bst 2.0 WarmStart® and WarmStart RTx for amplification of RNA and DNA/cDNA targets and a 50X LAMP Fluorescent Dye that binds to dsDNA for real-time detection. The WarmStart Fluorescent LAMP/RT-LAMP Kit (with UDG) contains a master mix that is formulated with 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). Q: What components are included in the WarmStart Fluorescent LAMP/RT-LAMP Kit (with UDG)? A: The kit includes a 2X Master Mix containing a reaction buffer with all necessary cofactors at optimized 2X concentrations for LAMP assays: dNTPs, MgSO4, Bst 2.0 WarmStart® DNA Polymerase, WarmStart RTx, Antarctic Thermolabile Uracil DNA Glycosylase (UDG), and dTTP/dUTP. The kit also contains a 50X LAMP Fluorescent Dye that binds to dsDNA for real-time detection of LAMP. To perform a LAMP reaction, the user adds primers, target DNA or RNA and adjusts the Master Mix to a final concentration of 1X. Detailed instructions can be found in the Kit Manual. Q: Does the WarmStart LAMP/RT-LAMP 2X Master Mix (with UDG) enable carryover prevention/contamination reduction? A: Yes, WarmStart LAMP/RT-LAMP 2X Master Mix (with UDG) is formulated with a mixture of dTTP and dUTP. This ensures both efficient isothermal amplification as well as the incorporation of dU into the reaction products. The mix also contains Antarctic Thermolabile UDG (NEB #M0372 ). LAMP products containing dU serve as a substrate for uracil DNA glycosylase, allowing carryover contamination prevention. Reactions should be set up at room temperature (typically 22-25°C) prior to isothermal incubation to allow dU-containing amplicons to be degraded, or if desired, a 10 minute, 25°C incubation before LAMP can be added to the workflow. Because LAMP can generate large quantities of DNA in very short periods of time, best practices to reduce contamination involve not opening LAMP reactions post amplification. 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: What are the advantages of using standard LAMP (NEB #M1712, E1700, M1708, E1708) over pH-based colorimetric LAMP (NEB #M1800, M1804)? A: Our pH-based colorimetric LAMP master mixes (NEB #M1800, NEB #M1804) utilize a weakly buffered solution to allow for visual detection using a pH-sensitive dye. This simple visual readout can be particularly useful for point of need testing. However, the low buffering capacity required to trigger the pink to yellow color change limits sample compatibility, as highly buffered sample inputs or acid samples may impact the color change. The master mixes in our standard LAMP products (NEB #M1712, NEB #E1700, NEB #M1708, NEB #E1708) can more readily tolerate these types of sample inputs. These products are also compatible with non-pH-based colorimetric detection. NEB’s LAMP/RT-LAMP master mixes enable choices regarding sample type and visual color change 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 phenol red, which is a pH-sensitive dye. The low buffering capacity permits the reaction pH to decrease as protons are produced from amplification of the target nucleic acid, generating a pink to yellow color change. This simple visual readout can be particularly useful for point of need testing. However, the low buffering capacity required for pH-based detection limits sample compatibility with the pH-based colorimetric LAMP mixes. Highly buffered sample inputs may inhibit the color change while acidic samples may sufficiently decrease the reaction pH to impact the initial color. 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 other colorimetric dyes (e.g., hydroxynaphthol blue, a metal indicator). 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.