New England Biolabs, M0208S, Taq DNA Ligase

DNA Ligase has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10233580. Related Categories DNA Ligases Applications Gibson Assembly,, Cloning Ligation Specification Unit Definition (Cohesive End Unit) One unit is defined as the amount of enzyme required to give 50% ligation of the 12-base pair cohesive ends of 1 µg of BstEII-digested λ DNA in a total reaction volume of 50 μl in 15 minutes at 45°C. Reaction Conditions 1X Taq DNA Ligase Reaction Buffer Incubate at 45°C 1X Taq DNA Ligase Reaction Buffer 20 mM Tris-HCl 25 mM Potassium Acetate 10 mM Magnesium Acetate 1 mM NAD 1 10 mM DTT 0.1% Triton® X-100 (pH 7.3 @ 25°C) Usage Concentration 40,000 units/ml Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No Unit Assay Conditions 1X Taq DNA Ligase Reaction Buffer and DNA (20 µg/ml). After incubation at 45°C for 15 minutes, the reaction is terminated by addition of stop dye (50% glycerol, 50 mM EDTA and bromophenol blue), heated at 70°C for 10 minutes and then loaded on a 0.7% agarose gel. Due to the presence of ligase, the cos ends of BstEII-digested λ DNA will stay together after 70°C heat treatment. FAQ Q: Is Taq DNA Ligase used for a special technique? A: Yes. It is used in LCR (ligase chain reaction) and LDR (ligase detection reaction). In general a single base change can be detected by two probes that meet with the terminal base of one probe pairing with the base being analyzed. If the terminal base pairs with the template, ligase can join the two probes. If the terminal base does not pair, ligase will not join the probes. Since the ligase is thermostable the reaction can be cycled and the ligation products amplified. Q: How much ligation occurs at mismatches when using Taq DNA Ligase? A: The maximum seen is 1.3 % ligation at mismatches. Q: How many temperature cycles will Taq DNA Ligase survive? A: At least 30 cycles. Q: Can Taq DNA Ligase be used for cloning? A: Taq DNA Ligase will not ligate all types of ends.We recommend using T4 DNA Ligase (NEB# M0202) for cloning. Q: What is the molecular weight of Taq DNA Ligase? A: The adenylated form is 77.2kD and the nonadenylated form is 76.9 kD. Q: Why is the Taq DNA Ligase buffer brown? A: The DTT and NAD react and turn brown if the buffer is stored at temperatures above -70°C. The brown color change does not affect enzyme activity. Q: Does Taq DNA Ligase require NAD? A: Yes. There is enough NAD bound to the enzyme so it will work to some extent in buffers that do not contain NAD. Q: What is the activity of Taq DNA Ligase in other NEBuffers? A: The activity of Taq DNA Ligase in other buffers is as follows: NEBuffer™ r2.1: 10,000 units/ml NEBuffer™ r3.1: 8-10,000 units/ml rCutSmart™ Buffer: 12-16,000 units/ml Full activity is achieved using unique Taq DNA Ligase Buffer, supplied with the enzyme: 40,000 units/ml. Q: What is the activity of Taq DNA Ligase at various temperatures? A: 4°C: 2,000 units/ml 16°C: 8-10,000 units/ml 25°C: 12,000 units/ml 37°C: 20,000 units/ml 45°C: 40,000 units/ml 55°C: 40,000 units/ml 65°C: 64,000 units/ml 75°C: 10,000 units/ml 85°C: 3-4,000 units/ml 95°C: < 500 units/ml Q: What is the stability of Taq DNA Ligase at 95°C? A: 0 min: 64,000 units/ml 1 min: 40-50,000 units/ml 5 min: 40-50,000 units/ml 10 min: 32,000 units/ml 15 min: 32,000 units/ml 20 min: 32,000 units/ml 30 min: 32,000 units/ml 45 min: 16,000 units/ml 60 min: 16,000 units/ml Q: What is the stability of Taq DNA Ligase at room temperature? A: Over one week. 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: 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: 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”. 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.