Description
T4 RNA Ligase catalyzes the ATP-dependent ligation of single-stranded nucleic acids (RNA or DNA) by joining a 5' phosphoryl-terminated nucleic acid donor to a 3' hydroxyl-terminated nucleic acid acceptor through the formation of a 3'→5' phosphodiester bond (1). Supplied in: 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol (pH 7.5 at 25°C) Supplied with: 10X T4 RNA Ligase Buffer (B6050): 500 mM Tris-HCl, 100 mM MgCl 2, 10 mM ATP, 100 mM DTT (pH 7.8 at 25°C)
Storage temperature: –25°C to –15°C Molecular weight: 43.5 kDa Test Amount tested Specification Purity n/a >99% Specific activity n/a 16,800 U/mg Single-stranded exonuclease 200 U <5.0 % released Double-stranded exonuclease 200 U <1.0 % released Double-stranded endonuclease 200 U No conversion E. coli DNA contamination 200 U <10 copies RNase contamination 200 U No detectable non-specific RNase
The enzyme is produced by a recombinant E. coli strain carrying the T4 RNA Ligase gene from bacteriophage T4. One unit is defined as the amount of enzyme required to ligate 50% of 0.4 μg of an equimolar mix of two single-stranded 23 base RNA oligonucleotides (one 5′-phosphorylated) in 20 μL 1X T4 RNA Ligase Buffer following a 30-minute incubation at 37°C.
Usage Instructions Single-stranded RNA circularization Set up the following reaction mixture in a total volume of 20 µL: Components Final Concentration Volume Nuclease-free water N/A X µL 10X T4 RNA Ligase Buffer (B6050) 1X 2 µL ssRNA with 5’P and 3’OH ends 200ng - 1µg X µL RNase Inhibitor (Y9240) 20 U 0.5 µL T4 RNA Ligase 1 (L6050L) 10 U 0.5 µL Total Volume = 20 µL Incubate at 25°C for 1—2 hours. Reaction can be stopped by adding EDTA to a final concentration of 12.5mM or clean-up using a spin column-based method. Notes: For hard-to-ligate single-stranded substrates, ligation efficiency can be improved by adding PEG at a final concentration of 5—10%. For longer single-stranded RNA substrates, overnight incubation at 16°C can improve yield. Quality Control Unit activity is measured using a 2-fold serial dilution method. Dilutions of the enzyme were made in 1X T4 RNA Ligase reaction buffer and added to 20 µL reactions containing 0.4 µg of an equimolar mix of two single-stranded 23 base RNA oligonucleotides (one 5′-phosphorylated) and 1X T4 RNA Ligase Buffer. Reactions were incubated for 30 minutes at 37°C, stopped and analyzed on a 15% TBE-Urea gel stained with SYBR® Gold Nucleic Acid Gel Stain (Invitrogen S-11494). Protein concentration (OD 280) is determined by OD 280 absorbance. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample. Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C. E. coli 16S rDNA Contamination is evaluated using 5 µL replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. Non-specific RNase contamination is assessed using the RNase Alert kit (Integrated DNA Technologies), following the manufacturer’s guidelines.
