Typhoon fla 9500 phosphorimager

For most experiments, cells were cultured in YEA medium that contains phosphate. In experiments comparing levels of pho1 expression in the presence or absence of phosphate, EMM was used with or without 15.5 mM sodium phosphate and 20 mM potassium phosphate. Total RNA was isolated by incubating cells in hot phenol heated to 65 °C for 10 min followed by 3 additional extractions using phenol-chloroform. RNA was precipitated using the sodium-acetate-ethanol method. Northern blots were performed according to the published protocol^{6 (link)}. 10 µg of RNA was resolved on a 1% formaldehyde-agarose denaturing gel and capillary transferred using NorthernMAX transfer buffer (Thermo Fisher Scientific) onto positively charged BrightStar-Plus nylon membrane (Ambion) and crosslinked using UV Stratalinker 2400 (Stratagene). The T7 in vitro transcription kit (Promega) was used to generate α-P³²-UTP (PerkinElmer) labeled RNA probes (Supplementary Table 2) that were hybridized to the membrane overnight at 65 °C in ULTRAhyb buffer (Ambion). The membrane was exposed and scanned using a Typhoon FLA 9500 phosphor imager (GE Healthcare).

APPXL-containing substrates were assembled by annealing the 40-mer template strand with the 11-mer [³²P]-labeled primer (run_pri) and, if necessary, with the downstream strand (28down), as described in [91 (link),92 (link)]. The cross-link was placed in either the template or the downstream strand. To prepare the AP-site-containing substrates, oligonucleotides were assembled in the same way, but with a uracil-containing 40-mer template strand (40U), and treated with Ung for 20 min at 37 °C immediately before the reaction. The reaction mixture (40 μL) contained 50 nM DNA substrate, dNTPs (each at 250 μM) and DNA polymerase in the appropriate reaction buffer (Table 3). The reaction was allowed to procced at 37 °C; aliquots were withdrawn at 2, 5 and 30 min, mixed with an equal volume of the stop solution (80% formamide, 20 mM Na-EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue) and heated for 2 min at 95 °C. The reaction products were separated via electrophoresis in 20% denaturing polyacrylamide gel and visualized using a Typhoon FLA 9500 phosphorimager (GE Healthcare, Chicago, IL, USA). Quantity One v4.6.8 (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify the band intensity.

Leading-strand replication assays were set up in eukaryotic replication (ER) buffer (25 mM Tris-OAc pH 7.5, 5% glycerol, 80 μg/mL BSA, 5 mM tris(2-carboxyethyl)phosphine, 10 mM Mg(OAc)₂, 50 mM potassium glutamate, 0.1 mM EDTA), and contained 1.5 nM DNA substrate (see section on Forked Linear DNA substrates), 30 nM CMG, 30 nM MTC, 20 nM Pol ε, 10 nM RFC, 30 nM PCNA, 600 nM RPA, 5 mM ATP and 120 μM dNTPs, and where indicated 40 nM sgRNA and 20 nM dCas9-dL5 in a final volume of 20 μL. First, DNA was incubated with CMG and MTC for 2 min at 30 °C followed by an additional 2 min with dCas9-dL5 and cgRNAs. Components except ATP and RPA were added and further incubated for 5 min at 30 °C. Replication was initiated by addition of ATP and RPA. The reactions proceeded for the indicated amount of time at 30 °C and were quenched with an equal volume of 2x stop solution (40 mM EDTA and 2% (w/v) SDS). DNA products were separated on a 1.3% (w/v) alkaline agarose gel at 35 V for 16 h. Gels were backed with DE81 paper (GE Healthcare), dried by compression, exposed to a phosphorimager screen, and imaged with a Typhoon FLA 9500 PhosphorImager (GE Healthcare).

Plantlets pretreated
with metcamifen or benoxacor as detailed above
were transferred into fresh media containing 25 μM [phenyl-U-¹⁴C]-mesotrione (4.07 MBq mg^–1, >90% pure).
Plantlets were harvested (n = 3) at 4.5, 24, 48,
and 72 h. The roots were washed with acetonitrile to remove unabsorbed
radioactivity and then separated into root, seed, stem, and leaf tissues.
Tissues were flash frozen before being pulverized in liquid nitrogen.
Finely ground tissues were extracted once with 2 v/w acetonitrile:water
(4:1 v/v) and then with 2 v/w acetonitrile:water (1:1 v/v). Combined
solvent extracts (50 μL) were radioassayed by liquid scintillation
counting (5 mL Prosafe+, TrisKem International, Bruz, France) and
then applied onto TLC silica gel 60 F254 plates (Sigma-Aldrich), after
standardizing the dpm applied. TLC plates were developed using chloroform:ethyl
acetate:methanol:formic acid (30:20:20:2 (v/v)), with cochromatographing
reference metabolites visualized under UV light and radioactive metabolites
quantified on a Typhoon FLA 9500 phosphorimager (GE Healthcare, Amersham,
UK, Multiguage V2.2 software).

DNA was extracted from 3,000 flow-sorted T cells per subset using a QIAmp DNA Micro Kit (Qiagen). STELA was carried out at the XpYp and 17p telomeres as described previously (25 (link)). For each sample, 1 µM of the Telorette-2 linker was added to purified genomic DNA in a final volume of 40 µL. Multiple PCRs were performed for each test DNA in volumes of 10 µL incorporating 1 µL of the DNA/Telorette-2 mix and 0.5 µM of the telomere-adjacent and Teltail primers in 75 mM Tris-HCl pH 8.8, 20 mM (NH₄)₂SO₄, 0.01% Tween-20, and 1.5 mM MgCl₂, with 0.5 U of a 10:1 mixture of Taq (ABGene) and Pwo polymerase (Roche Molecular Biochemicals). The reactions were processed in a Tetrad2 Thermal Cycler (Bio-Rad). DNA fragments were resolved using 0.5% Tris-acetate-EDTA agarose gel electrophoresis and identified via Southern hybridization using a random-primed α-³³P-labeled (PerkinElmer) TTAGGG repeat probe, together with probes specific for the 1 kb (Stratagene) and 2.5 kb molecular weight markers (Bio-Rad). Hybridized fragments were detected using a Typhoon FLA 9500 Phosphorimager (GE Healthcare). The molecular weights of the DNA fragments were calculated using a Phoretix 1D Quantifier (Nonlinear Dynamics).

Reactions were done as previously described^{79 (link),80 (link)} with some modifications. Briefly, reactions (10 µl) were done in BER buffer (40 mM HEPES, pH 7.6, 0.1 mM EDTA, 5 mM MgCl₂, 0.2 mg/ml BSA, 50 mM KCl, 1 mM DTT, 40 mM phosphocreatine, 100 µg/ml creatine phosphokinase (Sigma-Aldrich, USA), 2 mM ATP (GE-Healthcare, USA), 40 µM of each dATP, dTTP, dGTP and 4 µM of dCTP (Roche Applied Sciences, USA), 2 µCi ³²P-dCTP (Perkin Elmer, USA), 3% glycerol, and 100 fmol of uracil-containing double-stranded DNA substrate). Reactions were initiated by adding 0.4 µg whole tissue lysate and were incubated at 37 °C for 1 h, followed by termination with the equal volume of formamide stop dye (90% formamide, 10 mM EDTA, 0.01% bromphenol blue, 0.01% xylene cyanol). Samples were heated at 37 °C for 10 min and then run on 20% denaturing polyacrylamide gel (PAGE-Urea). The reactions were visualized using a Typhoon FLA 9500 PhosphorImager and quantitated with ImageQuant TL v8.1 software (GE Healthcare). The experiments were performed at least in duplicate. The incorporation, ligation and incorporation plus ligation activities are presented as PhosphorImager units per min per μg protein.