T4 RNA Ligase 1 (C007B-585734)

Storage Buffer： 10mM Tris-HCl, 50mM KCl, 0.1mM EDTA, 1mM DTT, 50% Glycerol, pH 7.5  T750872Component600U3000 UStorageT750872AT4 RNA Ligase 120 μL100 μL-20°C. Avoid freeze/thaw cycleT750872B10×T4 RNA Ligase 1 Buffer100 μL500 μL-20°C. Avoid freeze/thaw cycleT750872C50% PEG 8000100 μL500 μL-20°C. Avoid freeze/thaw cycleT750872DATP10 μL50 μL-20°C. Avoid freeze/thaw cycleT4 RNA Ligase 1 is an ATP-dependent single-stranded nucleic acid molecule (DNA or RNA) ligase that catalyzes the 5' phosphate group of the donor and the 3' hydroxyl group of the ligand to form 3'→5' phosphodiester bonds and accompanies ATP hydrolysis to AMP and PPi. The substrates of this enzyme are ssRNA, ssDNA and dinucleoside pyrophosphate.Enzyme Activity DefinitionAt 37℃, the amount of enzyme required to convert 1 nmol of 5 '-[32P] rA16 into phosphate insoluble matter within 30 min is defined as 1 unit (U).Protocol1. Cyclization of RNA(1) Prepare the mixed solution as shown in the following table:ComponentVolumessRNA200 ng-1 μg10×T4 RNA Ligase 1 Buffer2 μlATP20-50 μM50% PEG 800010% PEG 8000Murine RNase Inhibitor0.5-1 μlT4 RNA Ligase 11 μlNuclease-free WaterTo 20 μl(2) Incubation at 25 °C for 2h or 16°C for 16h.(3) The reaction was terminated by boiling at 65 °C for 15 min or 2min.2. Linking reaction between RNA or DNA oligonucleotides and ssRNA(1) Prepare the mixed solution as shown in the following table:ComponentVolumessRNA1-20 pmolRNA or DNA oligonucleotides5-40 pmol10×T4 RNA Ligase 1 Buffer2 μlATP2 μl50% PEG 800015% PEG 8000Murine RNase Inhibitor0.5-1 μlT4 RNA Ligase 11 μlNuclease-free WaterTo 20 μl(2) Incubation at 25°C for 2h or 16°C for 16h.(3) The reaction was terminated at 65°C15 min or by boiling for 2min.CautionsThis product is for scientific research purposes only and shall not be used for other purposes.

Specifictaions: EnzymoPure™, BioReagent, for DNA synthesis, Suitable for molecular biology, PCR Reagent, for DNA and RNA applications, 30 U/μl