New England Biolabs, M1708S, WarmStart® Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG)

Related Categories Isothermal Amplification & Strand Displacement,, Master Mixes 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: 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: 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: What detection methods can be used for LAMP/RT-LAMP experiments? A: The LAMP master mix is compatible with a variety of visualization/detection options including turbidity, fluorescence, non-pH-based colorimetric (e.g., hydroxynaphthol blue) and gel-based methods. The LAMP kits (NEB #E1700 and NEB #E1708) come with a 50X LAMP Fluorescent Dye that binds to dsDNA for real-time fluorescence detection. 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: 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.