T4 kinase

Various reagents used in this study were purchased from the following suppliers: IQ supermix, and Agarose from Bio-Rad; DNA purification Kit using Nucleospin Extract II columns from Machery Nagel, Germany (Cat no. 740609-50); T4 Kinase Polynucleotide from Invitrogen; γ-³²P- ATP from Amersham (3000 Ci/mmol); SSDNA from Sigma Aldrich cat# D7656; Sephadex columns from GE Healthcare (Microspin G-25 column illustra 27-5325-01); and T4-Kinase from Invitrogen, Life Technology.
DNA concentration was quantified using the Gene Quant Spectrophotometer. PCR was performed using My Cycler Thermal Cycler from Bio-Rad; Membrane hybridization and crosslinking were carried out using ProBlot 12 Hybridization Oven Labnet, (31 Mayfield Avenue Edison, NJ, 08837 USA), and Spectrolinker UV Crosslinkers from Krackeler Scientific, (Inc. PO Box 1849 Albany, NY 12201-1849), respectively. Sequencing of purified DNA was carried out at the University of Saint Joseph, Department of Molecular Biology and Genetics using the Avant Genetic Analyzer (ABI 3130) machine. The sequencing reaction and subsequent purification steps were as described before [9] (link).

Total RNA (15 μg) from samples were separated on 15% denaturing polyacrylamide gels, transferred onto GeneScreen Plus membranes (PerkinElmer), and hybridized using UltraHyb-Oligo buffer (Ambion). Oligonucleotides complementary to mature miR-130a (5’-AGCAAAAATGTGCTAGTGCCAAA-3’) were end-labeled with T4 Kinase (Invitrogen) and used as probes. Following hybridization at 42°C overnight, the membranes washed twice in 0.1 × SSPE and 0.1% SDS at 42°C for 15 min each. Membranes were then exposed to a storage phosphor screen (GE Healthcare Bio-Sciences) for 8 h and imaged using a Typhoon 9410 Variable Mode Imager (GE Healthcare Bio-Sciences). Northern blots hybridized with a 5S ribosomal RNA (rRNA) cDNA were used as controls.

Linear DNA containing the sabA promoter region (spanning −166 to +74) was generated by high-fidelity PCR using genomic DNA from SMI109 as template and primers sabA-5 and sabA-8. Radio-labeled DNA fragments were generated by first pre-labeling the sabA-5 primer using [γ³²P]ATP (>3000 Ci/mmol; Perkin Elmer) and T4 kinase (Thermo Scientific). The binding reactions with 10 nM DNA and increasing concentrations of E. coli σ⁷⁰-RNAP (Holoenzyme, Epicentre) were done as previously described [89] (link). The samples were separated on 4.5% Tris-Glycine (pH 8.5) polyarcylamide gel. The bands were visualized using Phosphor screen cassette, Typhoon scanner 9400 (GE Healthcare) and ImageJ software (NIH).

The oligonucleotides were end labeled using T4 kinase (ThermoScientific) and γ-ATP at 37°C and then purified on native polyacrylamide gels containing 10 mM NaCl (for RNA substrates) or KCl (for DNA substrates). EMSA was carried out in 20 μL reaction mixtures containing 0.4 nM ³²P-radiolabeled DNA/RNA, 50 mM Tris, 50 mM NaCl (for RNA substrates) or KCl (for DNA substrates), 2 mM MgCl₂, 2 mM DTT, 0.25 mg/ml BSA, and various concentrations of Oga1/Dbp2/Ded1 protein variants or mitochondrial single-stranded DNA binding protein (mtSSB). The mixture was incubated for 10 min at 30°C before adding 4 μL of 6× loading buffer (40% glycerol, 0.2% bromophenol blue, 60 mM EDTA). The samples were separated by electrophoresis on a 10% native acrylamide gel in Tris/Borate/EDTA buffer. The gel was then dried on Whatman paper, exposed overnight to a phosphorimager (Fujifilm), visualized with a Typhoon 9400 Variable Mode Imager (GE Healthcare), and quantified with the ImageQuant software. The helicase assay was performed similarly to EMSA with the exception that 1 mM ATP was included in the reaction mixture. The reaction was stopped by adding 4 μl of 6× helicase stop buffer (40% glycerol, 0.2% bromophenol blue, 60 mM EDTA, 0.6% SDS and 2 mg/mL proteinase K) before a further incubation of 10 min at 37°C.