Typhoon biomolecular imager

DFRSc was used in the reaction to measure the kinetics of amino acid activation. Reactions (final volumes of 20 µL) were performed at 37°C in 150 mM Na-HEPES (pH 7.2), 10 mM kF, 10 mM MgCl₂, 50 mM KCl, 0.2 mg/mL BSA, 10 mM DTT, 2 mM ATP, 1 mM PP_i, 0.2 µCi [γ-³²P]-ATP, 10 µM DFRSc and ncAAs 1-6 in different concentration ranges. Reactions were collected (1 µL for each) over time (0, 1, 2, 3, 4, 5 min) and spotted onto TLC PEI cellulose F plates (Merck). TLC plates were developed in a running buffer (1 M KH₂PO₄ and 4 M urea, pH 3.5) for approximately 25 min to separate ATP and PP_i. The air-dried plates were exposed on image plates, scanned on Amersham Typhoon Biomolecular Imager, and quantified using ImageJ software (Guo et al., 2014 (link)).

l-DNA aptamers isolated by DGGE were amplified by mirror-image PCR using d-Dpo4-5m in four separate reactions, in which one of the l-dNTPs was replaced by the corresponding l-dNTPαS (ref. ^{14 (link)}), with 5′-FAM-labeled l-DNA forward sequencing primer and unlabeled l-DNA reverse primer listed in Supplementary Table 6. The 5′-FAM-labeled PCR products were purified by 10% denaturing PAGE in 7 M urea and dissolved in 10 mM Tris-HCl at pH 7.4 to a final concentration of roughly 20 ng μl⁻¹. For each sequencing reaction, 5 μl of 5′-FAM-labeled l-DNA was mixed with 5 μl of cleavage solution containing 2% (v/v) 2-iodoethanol in ddH₂O, before being heated to 95 °C for 3 min and quickly placed on ice. For the removal of 3′-monophosphate from the 2-iodoethanol-cleaved DNA fragments, each sequencing reaction was treated with 5 units of natural CIP, incubated in 1× CutSmart buffer at 37 °C for 1 h, before being mixed with 10 μl of 2× loading buffer containing 95% formamide and 10 mM EDTA. The samples were loaded on slabs of 0.4 × 340 × 300 mm, analyzed by 10% denaturing PAGE in 7 M urea as described in the literature^{14 (link)} and scanned by the Amersham Typhoon Biomolecular Imager under the Cy2 mode. Chromatogram analysis was performed by the ImageJ software and Microsoft Excel.

TectoRNA assemblies were probed using a lead acetate cleavage assay as described previously (33 (link),38 (link),51 (link)). For each assembly, unlabeled tectoRNA at various concentrations ranging from 5 nM to 20 μM was mixed with ∼1 nM of 3′-[³²P]pCp labeled tectoRNA to monitor assembly and cleavage. For the cleavage assays, tectoRNA assemblies were first denatured at 95°C for 1 min and immediately cooled on ice for 2 min. Using a thermocycler, each tectoRNA sample was incubated at 20°C for 5 min before the addition of association buffer containing 25 mM HEPES pH 7.5, 50 mM KOAc, and either 0.05 mM, 0.5 mM or 2 mM Mg(OAc)₂ in the presence or absence of 200 μM folinic acid (FA). Following a 30-min incubation period at 20°C, 1 μl of 10 mg/ml tRNA was added prior to addition of 10 mM (final) Pb(OAc)₂. After 5 min at 20°C, the reaction was quenched with 10 μl of 100 mM EDTA solution. Each sample was ethanol precipitated, washed, and resuspended in colorless loading buffer (10 M urea, 1.5 mM EDTA) prior to loading onto a 10% (19:1) denaturing PAGE gel. Labeled RNA molecules were visualized using a phosphor-screen and a Typhoon Biomolecular Imager (Amersham). Lane profiles and densitometry analysis were analyzed using ImageJ 2.0 software.

Cells in supplemented EMM were collected and whole-cell protein extract was prepared by vortexing acid-washed glass beads in 20% trichloroacetic acid (TCA) and washing beads with 5% TCA. Lysates were boiled for 5 min in Laemmli Sample buffer (4% SDS, 60 mM Tris-HCl, pH 6.8, 5% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) and analyzed by SDS-PAGE. Primary antibodies used were as follows: anti-phospho-H2A (Abcam ab17353; 1:1,000), anti-H2A (Cell Signaling 3636S; 1:1,000; re-probed after phospho-H2A), anti-GFP (Abcam ab291; 1:1,000), anti-myc (Abcam ab9106; 1:1,000) anti-PCNA (Santa Cruz sc-56; 1:1,000), and anti-cdc2 (Abcam ab5467; 1:1,000). After secondary antibody (Alexa Flour 488 or 647; 1:6,000) incubation, blots were developed using Amersham Typhoon biomolecular imager. Intensity of bands were quantified with ImageJ.

RNA was isolated from strains HV30 × pTA232-tele-0582anti#1, -#2, and -#3 and wild-type strain HV30 × pTA232 as described before (64 (link)). Ten micrograms of RNA was separated on a denaturing 8% polyacrylamide gel or a denaturing 1% agarose gel and transferred to a nylon membrane (Hybond-N⁺ membrane or Pall membrane). For hybridization experiments, radioactively labeled PCR probes against the desired targets were generated using the DECAprime II DNA labeling kit and [α-³²P]dCTP (Life Technologies, Thermo Fisher Scientific). Membranes were subsequently incubated with the labeled PCR fragments.
To quantify the repression efficiency, Northern blotting membranes were exposed to imaging plates and analyzed with the Amersham Typhoon biomolecular imager and ImageQuantTL software. Signals were compared with the signals for the 16S rRNA used as a loading control. The amount of RNA signals detected for the wild-type controls was set to 100%. Northern blot analyses were done in triplicates.