New England Biolabs, M0328S, Bst DNA Polymerase, Full Length

Optimal performance in a number of applications Related Categories Isothermal Amplification & Strand Displacement Applications Isothermal Amplification Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 65°C. Reaction Conditions 1X ThermoPol® Reaction Buffer Incubate at 65°C 1X ThermoPol® Reaction Buffer 20 mM Tris-HCl 10 mM (NH4)2SO4 10 mM KCl 2 mM MgSO4 0.1% Triton® X-100 (pH 8.8 @ 25°C) Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% Triton® X-100 pH 7.1 @ 25°C Heat Inactivation 80°C for 20 minutes 5' - 3' Exonuclease Yes 3' - 5' Exonuclease No Strand Displacement No Unit Assay Conditions 50 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM MgCl 2 , 30 nM M13mp18 SS DNA, 70 nM M13 sequencing primer (–47) 24-mer, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 100 µM dTTP including [ 3 H]-dTTP and 100 µg/ml BSA. FAQ Q: What are Hot Start and WarmStart® polymerases and when would I use them? A: When setting up room temperature reactions off ice When non-specific amplification is observed What does Hot Start/ Warm Start mean? Our Hot Start and WarmStart polymerases utilize aptamers that inhibit enzyme activity at room temperature, which discourages the formation of nonspecific products. The presence of these aptamers does not alter core enzyme function. The distinction between Hot Start and WarmStart is that Hot Start enzymes are thermophilic while WarmStart® enzymes are mesophilic; the thermodynamic range of the aptamers for Hot Start and WarmStart enzymes are largely similar. Aptamers are engineered oligonucleotides that bind to a specific target molecule through non-covalent interactions and include specific nucleobase modifications that can improve inhibition profiles and/or reduce unintended side effects. The benefit of aptamer-based inhibition over other Hot Start technologies (like antibodies) is that they do not require an activation step and bind reversibly in a temperature-dependent manner. NEB offers polymerases with aptamers for routine PCR, high-fidelity PCR, isothermal amplification, and reverse transcription. These aptamers can target different enzymatic functions for different polymerases. Learn More Why is it called "Hot Start?" Like many non-proofreading, Family A DNA polymerases, Taq Polymerase possesses the ability to add bases onto the end of ssDNA in a template-independent manner even at room temperature, and can result in the addition of non-specific bases onto the ends of DNA primers in the reaction, enabling off-target hybridization and reduced overall reaction specificity. In contrast, at higher temperatures, nonspecific binding is reduced as annealing becomes more stringent. Early methods to mitigate undesired activity at low temperature focused on the exclusion of key reaction components until the reaction temperature was increased, which could then be spiked into the mixture, triggering the reaction under a more restrictive, high temperature condition. Read our feature article, Using aptamers to control enzyme activities Hot Start Taq and beyond, to see data comparing product formation for enzymes with and without aptamer-based inhibition of activity.