New England Biolabs, L4401P, LyoPrime WarmStart® Fluorescent LAMP/RT-LAMP Mix (with UDG)

Related Categories Isothermal Amplification & Strand Displacement Applications Loop-Mediated Isothermal Amplification,, Isothermal Amplification 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: 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: 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: 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: 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. 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 is the concentration of the LAMP Fluorescent Dye in the LyoPrime WarmStart Fluorescent LAMP/RT-LAMP Mix (with UDG)? A: LyoPrime WarmStart Fluorescent LAMP/RT-LAMP Mix (with UDG) contains LAMP Fluorescent Dye (NEB #B1700) at 0.5X concentration at 1X final reaction. The fluorescent dye can be detected using the SYBR®/FAM channel of common real-time PCR instruments. Q: How should the LyoPrime LAMP mix be stored? A: Vial format: Prior to use, LyoPrime LAMP products should be stored at room temperature (15 to 25°C), protected from light (e.g., in the original product box or another closed box). The glass vial, cap and rubber stopper are impermeable to moisture, allowing storage without desiccant. Storage at -20°C is not necessary and not recommended prior to rehydration. Additional storage and rehydration information can be found here. Plate format: Prior to use, the LyoPrime LAMP Mix should be stored at room temperature (15°C to 25°C) unopened in its original foil pouch. The foil pouch and desiccant inside protect the lyophilized reagent from atmospheric moisture and light. Any unused strip tubes should immediately be placed back in the foil pouch with the desiccant, sealing the zip-lock well and storing at room temperature. Additional storage and rehydration information can be found here. Q: How do I rehydrate the LyoPrime LAMP mix? Can it be resuspended at a higher concentration? A: Vial format: To rehydrate the lyophilized mix as a 2X Master Mix, remove the vial cap and stopper, add 675 μl nuclease-free water, and briefly mix by pipetting or gentle vortexing. The lyophilized mix can be rehydrated at up to 4X to accommodate higher sample volumes. However, reconstitution at higher concentrations than 2X may negatively impact time to detection for some targets. Please also see the protocol for additional information. Plate format: The lyophilized mix in each well can be rehydrated directly with primers and template to achieve 1X Master Mix (25 μl reaction volume). Therefore, it is highly recommended to prepare an assay mix including primers that is dispense into each well. Following adding assay mix, briefly mix by pipetting or vortexing using microplate shaker and then add template. Rehydration information can be found on the protocol page here and is also demonstrated in the product overview video. Q: When I dissolve the lyophilized product, the solution at first appears turbid. Is this normal? A: Yes, this is normal. The lyophilized cake in vial format dissolves immediately, but the release of air from the cake results in a temporary increase turbidity. The air will naturally degas from solution over ~20 seconds or after gentle vortexing. In the 96-well plate-based format, gentle mixing or vortexing results in immediate dissolution of the cake. Q: For L4401P - Do I need to use the entire LAMP plate in a single experiment? How are the 8-well strip tubes separated? A: The 96 well plate and optical lids are provided in a tear-off format allowing use of the entire plate directly or partially. The twelve 8-well strips are partially attached to each other, so the required number of 8-well strip tubes can be separated by gently tearing away the desired number of strip tubes. The 8-well strips are clicked in the yellow shell frame and the yellow shell frame adaptor must be removed to separate one or more strip tubes. The 8-well strips and lids are labeled 1 to 12 at the bottom to provide a tracking system independent of the yellow shell frame adaptor. Please check Plate Information and Instrument Compatibility section in the protocol for more information. Q: For L4401P - How do I store unused 8-well strip tubes after opening the foil pouch? How long can I keep unused 8-well strip tubes? A: Any unused 8-well strip tubes should be immediately resealed in the original zip-lock foil pouch with the provided desiccant and can be stored at room temperature. It is recommended to use these tubes within 1 week of opening of the foil pouch for the best results. Q: For L4401P - How long can I keep the plate or the 8-well strip tubes out of the foil pouch before setting up the reaction? A: After taking the plate out of the foil pouch, it is recommended to hydrate the lyophilized reagent intended for use within 1 hour. Q: For L4401P - What is the purpose of yellow shell adaptor? A: The strip tubes are provided in a single use yellow shell frame semi-skirted adaptor. This yellow adaptor has chamfered edge at the upper left corner (A1), which supports the use of the product on ABI /Life Technologies® FAST Cyclers and robotic systems. The yellow shell frame provides rigidity to the plate, so it is highly recommended to use for full plate runs in the ABI /Life Technologies FAST Cyclers. If the plate will be used partially, individual 8-well strips can be used with or without the yellow shell frame in ABI /Life Technologies FAST Cyclers. Use of other instrument platforms (e.g., Bio-Rad®) will require removal of the yellow shell adaptor prior to thermocycling. Please see the Plate Information and Instrument Compatibility section in the protocol for recommendations on the use of the yellow shell adaptor in common real-time PCR instruments. Q: For L4401P - Can I reuse the yellow shell adaptor? A: The yellow shell adaptor is intended for single-use to prevent cross contamination between RT-qPCR runs. It should be discarded along with the strip tubes after the run is complete. Separating individual strips from the yellow shell adaptor post amplification may result in the unintended opening of the caps and potential amplicon contamination events. If additional yellow shell frame adaptors are needed, they can be purchased from BIOplastics (product code AB19805G.) Q: For L4401P - Why is there a cardboard tray in the package? A: The cardboard tray is included in the packaging to support the plate during shipping. It can be recycled upon opening the package, or it can be used as a support for the plate during room temperature setup.