{"product_id":"neb-m1712l","title":"New England Biolabs, M1712L, Bst-XT WarmStart™ Multi-Purpose LAMP\/RT-LAMP 2X Master Mix (with UDG)","description":"\u003cb\u003eRelated Categories\u003c\/b\u003e Isothermal Amplification \u0026amp; Strand Displacement  \u003cb\u003eApplications\u003c\/b\u003e Isothermal Amplification,, DNA Amplification, PCR \u0026amp; qPCR  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eMaterials Required but not Supplied\u003c\/b\u003e Heating Device: heat block, water bath, or real-time fluorimeter (qPCR instrument) set to 65°C  \u003cb\u003eFAQ\u003c\/b\u003e Q: What is the Mg++ concentration in the Bst-XT LAMP\/RT-LAMP Master Mix? A: The 2X Master Mix contains 14 mM MgSO4, thus 7 mM MgSO4 in the 1X final reaction. Q: What is the optimal LAMP\/RT-LAMP amplification temperature while using Bst-XT LAMP\/RT-LAMP Master Mix? A: We recommend running LAMP reactions at 65°C. However, it is possible to perform LAMP and RT-LAMP reactions from 50–70°C, depending on the nature of the primer set and target. Q: Does Bst-XT WarmStart Multi-Purpose LAMP\/RT-LAMP 2X Master Mix (with UDG) enable carryover prevention\/contamination reduction? A: Yes, Bst-XT WarmStart Multi-Purpose 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). In subsequent reactions, LAMP products containing dU serve as a substrate for UDG, uracil DNA glycosylase, allowing carryover contamination prevention. UDG activity during setup will quickly and efficiently destroy any contaminating products. Because LAMP can generate large quantities of DNA in very short periods of time, best practices to reduce contamination involve avoiding opening LAMP reactions after amplification. Q: How fast should I expect a result when using the Bst-XT LAMP\/RT-LAMP Master Mix? A: Target amplification will vary depending on a number of factors including template purity, template quantity, and primer design (we recommend the NEB LAMP Primer Design Tool). We suggest beginning with an incubation time of 20 minutes. If desired, the time can be shortened to 15 minutes with optimized assays or high copy targets. Alternatively, it can be lengthened to up to 40 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 do not become an issue. Q: What detection methods are compatible with the Bst-XT LAMP\/RT-LAMP Master Mix? A: The Bst-XT LAMP\/RT-LAMP Master Mix is compatible with a variety of visualization\/detection methods including fluorescence, turbidity, and end point colorimetric detection using certain metal-sensing dyes (e.g., calcein with manganese or Eriochrome Black T). This master mix is also compatible with gel-based evaluation and lateral flow methods. Please note that hydroxynaphthol blue is not recommended for use with this master mix because the resulting color change is not distinct. If the reaction will be monitored in real time, LAMP Fluorescent Dye (NEB #B1700) can be included at 0.5X final concentration to monitor the reaction in a SYBR\/FAM channel of real-time PCR instrument. The dye can be titrated as necessary between 0.1X and 1X. 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: 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: 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: 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: 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. 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