New England Biolabs, M0647S, HiFi Taq DNA Ligase

This product is available in a Related Categories DNA Ligases Applications Non-Cloning Ligation,, DNA Ligation Specification Reaction Conditions 1X HiFi Taq DNA Ligase Reaction Buffer 1X HiFi Taq DNA Ligase Reaction Buffer 20 mM Tris-HCl 150 mM KCl 10 mM MgCl2 10 mM DTT 1 mM NAD 0.1% Triton® X-100 (pH 8.5 @ 25°C) 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 No FAQ Q: What applications are HiFi Taq DNA Ligase best used for? A: HiFi Taq DNA Ligase is designed for use in methods of SNP detection by ligation, for example, LCR (ligase chain reaction) and LDR (ligase detection reaction). In these methods, the presence of a particular base at a single site can be detected through probes that meet with the terminal base of one probe pairing with the base being analyzed. If the terminal base forms a Watson-Crick base pair with the template at this position, the ligase can join the two probes. If the terminal base does not pair, the ligase will not join the probes. Since the ligase is thermostable the reaction can be cycled and the ligation products amplified. For more information regarding ligase specificity and high fidelity ligases, visit “Substrate specificity and mismatch discrimination in DNA ligases”. Q: Can HiFi Taq DNA Ligase buffer be used with other DNA ligases? A: Yes, HiFi Taq DNA Ligase buffer can be used with Taq DNA Ligase. Use of the HiFi Taq DNA Ligase buffer with Taq DNA Ligase will result in a modest fidelity and activity increase. ATP-dependent ligases (for example, 9°N™ DNA Ligase NEB #M0238 , T4 DNA Ligase NEB #M0202 and T7 DNA Ligase NEB #M0318 ) are incompatible with this buffer. For more information regarding ligase specificity and high fidelity ligases, visit “Substrate specificity and mismatch discrimination in DNA ligases”. Q: Can HiFi Taq DNA Ligase be used in other buffers? A: In order to achieve the highest fidelity possible with HiFi Taq DNA Ligase, we recommend using HiFi Taq DNA Ligase buffer, however, HiFi Taq DNA Ligase can be used in Taq DNA Ligase buffer with reduced activity and fidelity. HiFi Taq DNA Ligase can be used in a variety of polymerase buffers when supplemented with 1 mM NAD with reduced activity and modestly reduced fidelity. NEB Buffer HiFi Taq DNA Ligase Activity (%) Effect on Fidelity Hifi Taq DNA Ligase Buffer 100 - Taq DNA Ligase Buffer 38 ↓↓↓ phi29 Reaction Buffer1 54 ↓↓ Q5® Reaction Buffer1 73 ↓ Standard Taq Reaction Buffer1 90 ↓ ThermoPol Buffer®1 215 ↓↓ 1Supplemented with 1 mM NAD. 2Additional supplementation of polymerase buffers with 5 mM Mg may increase HiFi Taq DNA Ligase activity, but further reduce fidelity. Q: What is the optimal reaction temperature for HiFi Taq DNA Ligase? A: HiFi Taq DNA Ligase is active over a range of temperatures (35-75°C) with greatly increased activity at higher temperature up to the Tm of the probe oligonucleotides used. Typical ligations can be performed at 60°C. Ideally the reaction temperature should be chosen based on the Tm of the probes used, within a few degrees of the Tm of the probes used as calculated at the conditions of the buffer (10 mM MgCl2, 150 mM KCl) and the probe concentrations used. The optimal reaction temperature for a given application must be determined empirically. Q: How much HiFi Taq DNA Ligase is required for a reaction? A: A typical reaction uses 1 μL of HiFi Taq DNA Ligase in a 50 μL reaction volume. Please see the accompanying product information for details on reaction conditions including molar ratios or probes, target DNA, and enzyme. Q: How many temperature cycles will HiFi Taq DNA Ligase survive? A: HiFi Taq DNA Ligase remains over 97% active after more than 100 cycles (where 1 cycle = 80°C for 90 s, 94°C for 10 s). Q: Can HiFi Taq DNA Ligase be used for cloning? A: No, HiFi Taq DNA Ligase will not ligate all types of ends. We recommend using T4 DNA Ligase (NEB #M0202) for cloning. Q: Does HiFi Taq DNA Ligase require NAD+? A: Yes. Trace activity may be observed in buffers without NAD+ due to some cofactor pre-bound to the ligase. Q: What is the stability of HiFi Taq DNA Ligase at 95°C? A: The half-life of HiFi Taq DNA Ligase is greater than 90 min at 95°C. Q: What is the stability of HiFi Taq DNA Ligase at room temperature? A: Over one week. Q: Why is the HiFi Taq DNA Ligase Buffer brown? A: The DTT and NAD react and turn brown. The brown buffer will still work. Q: Do thermostable DNA ligases (such as Taq DNA Ligase, 9°N DNA Ligase, and HiFi Taq DNA Ligase) ligate sticky ends? A: While these thermostable ligases can join sticky ends with extensive overlap, like the 12 bp cohesive ends of the genome of bacteriophage Lambda, typical 4 bp overhangs generated by Type II restriction enzymes are not good substrates and will not be ligated. Thermostable DNA ligases are not recommended for use in traditional cloning workflows. Q: Does HiFi Taq DNA Ligase have activity on substrates containing RNA? A: HiFi Taq DNA Ligase has activity only on nicked DNA substrates with no mismatches or gaps. HiFi Taq DNA Ligase has no measurable activity on RNA or RNA/DNA nicked substrates. Q: What is LDR and how does it differ from LCR? A: LDR (Ligase Detection Reaction) is a ligation dependent methodology that, unlike LCR (Ligase Chain Reaction), involves only one pair of probes complementary to one strand of target DNA. Cycling in LDR results in linear amplification of the ligation product. This method can be used to confirm the presence of a particular SNP in a target sequence that has been amplified by another method (such as PCR). As with LCR, the method uses a high fidelity thermostable ligase that can discriminate against the ligation of mismatched probes, such as Taq DNA Ligase (NEB #M0208) and 9°N™ DNA Ligase (NEB #M0238). For more information regarding ligase specificity and high fidelity ligases, visit “Substrate specificity and mismatch discrimination in DNA ligases”. Q: What is LCR and which enzymes do you recommend? A: LCR (Ligase Chain Reaction) is a method similar to PCR (Polymerase Chain Reaction) that amplifies DNA and has promising diagnostic applications. LCR is a ligation-dependent method that can distinguish between DNA sequences differing in by only one nucleotide (SNPs). Four probes are used in LCR, one pair complementary to each strand in the target sequence, with the ligation junction at the SNP to be discriminated. The method uses thermostable DNA ligases such as Taq DNA Ligase (NEB #M0208) and 9°N™ DNA Ligase(NEB #M0238). For more information regarding ligase specificity and high fidelity ligases, visit “Substrate specificity and mismatch discrimination in DNA ligases”. Q: How can I design probes for LDR or LCR to maximize specificity? A: Probes for LDR (Ligase Detection Reaction) and LCR (Ligase Chain Reaction) should be designed so that all anneal within a narrow temperature range (ideally < 2°C), and should have annealing regions 15-30 bases long. Typically, the best reaction temperature for highest fidelity will be approximately between 2°C below and 2°C above the Tm for a given probe set, calculated according to the buffer conditions used. Use of the Thermostable Ligase Reaction Temp Calculator is highly recommended for selection of an incubation temperature. However, the optimal reaction temperature for a given probe set must be determined empirically. When using HiFi Taq DNA Ligase, the SNP to be discriminated can pair with either the 3´ base of the upstream probe (providing the 3′-hydroxyl to the ligation junction) or the 5′ base of the downstream probe (providing the 5´-phosphate to the ligation junction), as HiFi Taq DNA Ligase exhibits increased discrimination between correct and mismatched base pairs at either side of the ligation junction. Please see the accompanying product information for a detailed analysis of the fidelity of mismatch ligation by HiFi Taq DNA Ligase that illustrates its higher fidelity at both the 3´ and 5´ side of the ligation junction and specific base pair discrimination ratios. For more information regarding ligase specificity and high fidelity ligases, visit “Substrate specificity and mismatch discrimination in DNA ligases”.