{"product_id":"neb-m0238s","title":"New England Biolabs, M0238S, 9°N™ DNA Ligase","description":"9°N DNA Ligase has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10230152.  \u003cb\u003eRelated Categories\u003c\/b\u003e DNA Ligases  \u003cb\u003eApplications\u003c\/b\u003e Cloning Ligation  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eUnit Definition\u003c\/b\u003e (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. A cohesive end unit is equivalent to the nick-closing unit (1).  \u003cb\u003eReaction Conditions\u003c\/b\u003e 1X 9°N DNA Ligase Reaction Buffer Incubate at 45°C 1X 9°N DNA Ligase Reaction Buffer 10 mM Tris-HCl 600 µM ATP 2.5 mM DTT 2.5 mM MgCl2 0.1% Triton® X-100 (pH 7.5 @ 25°C)  \u003cb\u003eUsage Concentration\u003c\/b\u003e 40,000 units\/ml  \u003cb\u003eStorage Buffer\u003c\/b\u003e 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg\/ml Recombinant Albumin 50% Glycerol 10 mM ammonium sulfate pH 7.4 @ 25°C  \u003cb\u003eHeat Inactivation\u003c\/b\u003e No  \u003cb\u003eUnit Assay Conditions\u003c\/b\u003e 1X 9°N DNA Ligase Reaction Buffer and 20 µg\/ml BstEII-digested λ DNA in a 50 μl reaction. 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.  \u003cb\u003eFAQ\u003c\/b\u003e 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 \u0026lt; 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. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835520667817,"sku":"M0238S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0238s","provider":"Iright","version":"1.0","type":"link"}