New England Biolabs, M1202S, Tte UvrD Helicase

Related Categories PCR, qPCR & Amplification Technologies Applications Helicase-dependent Amplification,, Loop-Mediated Isothermal Amplification,, DNA Amplification, PCR & qPCR Specification Reaction Conditions 1X Isothermal Amplification Buffer Pack Supplement with 1 mM ATP Incubate at 65°C 1X Isothermal Amplification Buffer Pack 20 mM Tris-HCl 10 mM (NH4)2SO4 50 mM KCl 2 mM MgSO4 0.1% Tween® 20 (pH 8.8 @ 25°C) Usage Concentration 0.2 - 0.4 ng/μl Storage Buffer 10 mM Tris-HCl 100 mM KCl 1 mM DTT 0.1 mM EDTA 0.1% Triton® X-100 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 5 minutes FAQ Q: Does Tte UvrD Helicase (NEB #M1202) require any specific DNA structure for unwinding? A: No, Tte UvrD Helicase can utilize any dsDNA structure as a substrate for unwinding. This includes duplex DNA and duplexes with 3′and 5′ ssDNA overhangs (1-3). References An, L., Tang, W., Ranalli, T.A., Kim, H.J., Wytiaz, J., and Kong, H. (2005). J Biol Chem 280(32):28952-8. Mauris, J., and Evans, T.C. Jr. (2010). J Biol Chem 285(15):11087-92. Vincent, M., Xu, Y., and Kong, H. (2004). EMBO Rep 5(8):795-800. Q: How do I use Tte UvrD Helicase for reducing non-template amplification in LAMP reactions A: We suggest adding 10 ng Tte UvrD Helicase to a 25 μl LAMP reaction using the WarmStart® LAMP Kit (NEB #E1700). Helicase should be added to all reactions, as it has minimal effect on performance of reactions containing the target DNA or RNA (though fluorescence signal may decrease; reaction time should not be affected). For reactions with very high non-template amplification levels, the amount of helicase may need to be increased. If positive signal is affected, the amount of helicase should be reduced. Suggested ranges to use in LAMP reactions with indicated DNA polymerases are as follows: Bst DNA Polymerase, Large Fragment (NEB #M0275): 5-10 ng per reaction (0.2–0.4 ng/μl) Bst 2.0® DNA Polymerase (NEB #M0537): 10-15 ng (0.4–0.6 ng/μl) Bst 3.0 DNA Polymerase (NEB #M0374): 10–20 ng (0.4–0.8 ng/μl) Q: Is Tte UvrD Helicase compatible with the WarmStart Colorimetric LAMP 2X Master Mix (NEB #M1800)? A: No, Tte UvrD Helicase should not be used with Colorimetric LAMP. As the UvrD acts on DNA substrates it hydrolyzes dATP, releasing an H+ with each hydrolysis much like a DNA polymerase incorporating dNTP. This proton production will result in a drop in pH for the colorimetric LAMP solution independent of any amplification reaction, changing the color of all reactions containing the WarmStart Colorimetric LAMP 2X Master Mix and oligonucleotide primers. Q: Do I need to add ATP for Tte UvrD Helicase activity? A: If using Tte UvrD Helicase for helicase activity independent from any DNA polymerase or amplification activity, or in Isothermal Amplification Buffer, then yes, 1 mM ATP should be added to the reaction mixture. If adding Tte UvrD Helicase to an amplification reaction (for example, to WarmStart LAMP Master Mix) then the dATP present in the mixture will be utilized for unwinding. Q: Is Tte UvrD Helicase active in other buffers? A: Optimal activity is achieved with the recommended Isothermal Amplification Buffer supplemented with 1 mM ATP or in the WarmStart LAMP 2X Master Mix (NEB #E1700). But Tte UvrD Helicase is active in other buffers, and relative activity in some other potential buffers is shown below: Buffer Relative Activity NEBuffer 1.1 100% NEBuffer 2.1 100% NEBuffer 3.1 75% CutSmart® Buffer 100% Isothermal Amplification Buffer II 10% ThermoPol® Buffer 100% WarmStart LAMP Master Mix 100% Q: Can I purchase large amounts of an existing Enzyme for Innovation? A: Yes, if you have use for large amounts of an Enzyme for Innovation, please contact NEB at EnzymesForInnovation@neb.com. Please note that there may be limited availability and thus extended lead times required for large orders. Additional QC testing may also be necessary to meet your specific application and needs. Q: What are Enzymes for Innovation? A: Enzymes for Innovation (EFI) is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities that are not commercially available elsewhere at the quality that you would expect from NEB. Enzymes for Innovation graduates are enzymes that have transitioned from having unknown or exploratory applications to becoming the cornerstone of a defined application. If you have an idea for an “Enzyme for Innovation” with a suggested application that may be useful, please email us at EnzymesForInnovation@neb.com. For more information, please view video.