T4 DNA Ligase, Cloned

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always use the supplied buffer to avoid high salt concentration.	ATP should be added to the reaction to a final concentration of 0.5-1.0 mM.

Upstream tips
Always use the supplied buffer to avoid high salt concentration.
Protocol tips
ATP should be added to the reaction to a final concentration of 0.5-1.0 mM.

Publication protocol

Reactions were optimized progressively with respect to temperature and to the concentrations of ligase, ATP and oligonucleotide substrates. Temperature: Initial pilot reactions contained 17.5 µM radiolabeled dCHr8, 17.5 µM unlabeled capture oligo and 2.5 µM of each of seven bridging oligos (A0G, A1G, A2G, A4G, A6G, A8G, A10G). Oligo mixtures were denatured in water at 75°C for 3 min, then cooled on ice to anneal ligation complexes. Buffer was added to yield final concentrations of 1 mM ATP, 0.02 U/µL RNasin, 50 mM Tris pH 7.6, 66.7 mM KCl, 10 mM MgCl2 and 1 mM dithiothreitol. Ligation was initiated by addition of T4 DNA ligase to a final concentration of 6 µM ( = 0.33 µg/µL = 1U/µL) on ice, followed by overnight incubation at 10, 20 or 30°C. Reactions were stopped by adding an equal volume of gel-loading buffer (92% formamide, 20 mM EDTA, 0.01% bromophenol blue and 0.01% Xylene cyanol). Samples were separated on 10% denaturing polyacrylamide gels, which were dried and exposed to phosphorimager plates. Gel data were analyzed with ImageQuant software (Molecular Dynamics) by dividing the signal in each band (ligated product, adenylate, unreacted) by the total signal for all bands within the lane. Temperature was held at 20°C for all subsequent reactions. Ligation complex concentration: dCHr8, capture oligo and the A2G bridging oligo were mixed at ratio of 1∶1.25∶1.25, and assayed as above for ligation. Ligated product improved markedly at dCHr8 concentrations between 0.1 µM and 1.0 µM, with little further increase between 1.0 µM and 10 µM (Fig. S1B). Concentration of dXHr8 was held constant at 3.3 µM for subsequent reactions. Ligase concentration: Reactions were assembled for dCHr8/capture oligo/A2G bridge and incubated overnight (20 h) in various concentrations of T4 DNA ligase. The fraction ligated increased as enzyme concentration was increased from 0.06 to 6 µM (Fig. S1C). Yield approached saturation at 4 µM, which is the lowest concentration at which enzyme was in stoichiometric excess of dCHr8 (see Discussion). This concentration was used in subsequent reactions. ATP concentration: Finally, reactions were assembled for dCHr8/capture oligo/A2G bridge with added ATP concentrations ranging from 0 µM to 1000 µM, as detailed above (Fig. 3). For reactions carried out at 20°C, optimal joining was achieved with two non-annealed nucleotides separating the tethered mononucleotide from the annealed 8-mer, at low ATP concentrations (≤33 µM, to avoid enzyme re-adenylation) and high ligase concentrations (0.67 U/µL, in molar excess of annealed complexes), and at a ratio of 1∶1.25∶1.25 for the three nucleic acid strands (dXHr8: capture oligo: bridging strand).

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