New England Biolabs, M0375L, SplintR® Ligase

SplintR Related Categories RNA Ligation,, DNA Ligases Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) Unit Definition One unit is defined as the amount of enzyme needed to ligate (to 50% completion) 2 picomoles of a tripartite FAM-labeled DNA:RNA hybrid substrate in a 20 μl reaction at 25°C in 15 minutes in 1X SplintR Ligase Reaction Buffer. Reaction Conditions 1X SplintR Ligase Reaction Buffer Incubate at 25°C 1X SplintR Ligase Reaction Buffer 50 mM Tris-HCl 10 mM MgCl2 1 mM ATP 10 mM DTT (pH 7.5 @ 25°C) Storage Buffer 10 mM Tris-HCl 300 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes FAQ Q: Can you tell me what inhibits SplintR Ligase? A: SplintR Ligase is active between 4-37°C. Above this temp, the enzyme starts to denature. It is mostly inactivated after 10 minutes at 65°C. Additionally, monovalent cations such as NaCl and KCl inhibit the enzyme. These components should be kept below 50 mM in the reaction. We supply the enzyme in 300 mM NaCl to maintain robust activity over its shelf life at -20°C, but the enzyme needs to be diluted by addition to the reaction. This is easily achieved by choosing a 20-fold dilution in the reaction set-up (e.g. 1 ul of enzyme in a 20 ul reaction). As with most enzymes, the presence of ionic detergents and denaturants, such as SDS, urea, and Guanidine HCl should be avoided. Q: Can SplintR® DNA Ligase be used for ligation-based methods of microRNA detection? A: Yes, SplintR DNA Ligase has been show to be suitable for detection of microRNA by qPCR-coupled ligase detection reaction, and for detection using circularizable Padlock probes. SplintR Ligase overcomes the efficiency limitation of T4 DNA ligase for in vitro or direct in situ RNA detection by ligation, making SplintR Ligase an attractive tool for the detection and quantification of microRNA. Q: How can I maximize ligation fidelity when using SplintR DNA Ligase? A: Increasing reaction temperature has been shown to improve activity and specificity of SplintR DNA Ligase. Incubation at 37° C gives improved results in detection of microRNA. Addition of ET SSB (NEB #M2401S) has also been shown to increase activity and reduce off-target ligation when ligating DNA probes to RNA using SplintR DNA Ligase. Please see SplintR DNA Ligase product page for references. Q: I would like to use SplintR Ligase in a workflow, but not use the provided reaction buffer. Can you tell me what reaction buffer components are needed? A: We recommend a reaction with 20-50 mM buffering agent at a pH between 7.5-8.0. Additionally, magnesium should be present at ≥ 5mM for maximal activity. Manganese is not required but we observe an increase in activity when this divalent cation is present above 5 mM. ATP is necessary, but the enzyme is active over a broad range of concentrations (10 nM-1 mM). Q: SplintR Ligase seems very concentrated at 25 units per microliter. How do I know how much to add to my reaction? A: SplintR Ligase has been developed for the life sciences community, with a strong appreciation for its utility as a detection reagent. Some empirical optimization is likely necessary for individual applications. We employed a very sensitive assay, in which the substrate is present in nanomolar amounts. As such, the unit definition is targeted for applications where single nanogram amounts of substrate are routinely employed. The enzyme is supplied as a 10.5 uM solution. We suggest maintaining the enzyme below 1 uM in the reaction with a suggested range of 100 nM to 1 uM. For many applications, starting with a 2-fold excess of enzyme over ligatable ends is ideal. Additonally, if the reaction is not proceeding as efficiently as desired, we strongly recommend extending the incubation time rather than increasing the amount of enzyme in the reaction. Q: What is the salt tolerance of this RNA ligase? A: NEB # RNA Ligase Salt Tolerance M0204 T4 RNA Ligase 1 (ssRNA Ligase) ≤ 50 mM M0239 T4 RNA Ligase 2 (dsRNA Ligase) ≤ 50 mM M0242 T4 RNA Ligase 2, truncated ≤ 100 mM M0351 T4 RNA Ligase 2, trunc. K277Q ≤ 100 mM M0373 T4 RNA Ligase 2, trunc. KQ ≤ 100 mM M0375 SplintR Ligase ≤ 50 mM E2610 5' Adenylation Kit (Mth Ligase) ≤ 300 mM M0319 Thermostable 5' App DNA/RNA Ligase ≤ 50 mM Q: What type of ends does SplintR Ligase join? A: SplintR Ligase is extremely effective at sealing nicks between adjacent residues of DNA strands hybridized to RNA splints or bridges, with an efficiency often 10-100 times that seen for T4 DNA ligase. On a fully-DNA substrate, this nick-sealing activity is equivalent to other DNA ligases. It is not a good choice for molecular cloning workflows in which double-strand breaks need to be repaired (such as joining a vector and an insert).