Unless indicated otherwise, the experiments were performed in a 15-µL volume in 25 mM Tris-acetate (pH 7.5), 2 mM magnesium acetate, 1 mM ATP, 1 mM dithiothreitol, 0.1 mg/mL bovine serum albumin (New England Biolabs), 1 mM phosphoenolpyruvate, 16 U/mL pyruvate kinase (Sigma), and 1 nM (in molecules) 32P-labeled oligonucleotide-based, pUC19-based, or PCR-based DNA substrate or 0.15 nM λ DNA-based substrate (corresponding to 2.4 nM 5′-terminated ssDNA ends upon denaturation). For reactions with unlabeled substrates, 100 ng of DNA was used. Where indicated, human and yeast RPA was included to saturate all ssDNA. Recombinant proteins were added on ice to the assembled reaction mixtures. The reactions were incubated for 30 min at 30°C for yeast and 37°C for human proteins unless indicated otherwise. Reactions were stopped by adding either 5 µL of 2% stop solution for native gels as described previously (Cejka and Kowalczykowski 2010 (link)) or 15 µL of formamide dye for denaturing gels (Cannavo and Cejka 2014 (link)). All gels with radioactive substrates were dried on DE81 chromatography paper (Whatman) and exposed to storage phosphor screens (GE Healthcare). The screens were scanned by a Typhoon 9400 phosphorimager (GE Healthcare). Where unlabeled DNA substrates were used, DNA was visualized by staining with ethidium bromide (Sigma) or GelRed (Biotium) as indicated.
Typhoon 9400 phosphorimager
Manufactured by GE Healthcare
Sourced in United States
The Typhoon 9400 phosphorimager is a laboratory instrument designed for the detection and quantification of radioactively labeled biological samples, such as gels, membranes, and phosphor screens. It utilizes a laser-based scanning system to capture high-resolution digital images of the samples, allowing for accurate analysis of the radioactive signals.
Automatically generated - may contain errors
Lab products found in correlation
Molecular biology grade bovine serum albumin, by New England Biolabs (6 mentions)
Gotaq green, by Promega (4 mentions)
γ 32p atp, by PerkinElmer (4 mentions)
Rnase inhibitor, by New England Biolabs (4 mentions)
Avian myeloblastosis virus reverse transcriptase, by Promega (4 mentions)
Storage phosphor imaging plates, by GE Healthcare (4 mentions)
T4 polynucleotide kinase, by New England Biolabs (4 mentions)
Goscript reverse transcriptase, by Promega (3 mentions)
Dna oligonucleotide, by Integrated DNA Technologies (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
Rq1 rnase free dnase, by Promega (3 mentions)
Rna oligonucleotides, by GE Healthcare (3 mentions)
Deoxynucleotide triphosphate dntp mix, by Promega (3 mentions)
Dpn 1, by Thermo Fisher Scientific (3 mentions)
Storage phosphor screen, by GE Healthcare (2 mentions)
Quickchange 2 xl mutagenesis kit, by Agilent Technologies (2 mentions)
Ultraflextreme maldi tof tof mass spectrometer, by Bruker (2 mentions)
Gelred, by Biotium (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
19 protocols using typhoon 9400 phosphorimager
1
DNA Substrate Replication Assay
2
Quantifying Ush1c mRNA Expression
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One microliter of cDNA was used in PCR reactions with GoTaq Green (Promega) supplemented with primers and α–32P–dCTP. Primers specific for human Ush1c exon 3 (5′-GAATATGATCAGCTGACC-3′) and mouse exon 5 (5′-TCTCACTTTGATGGACACGGTCTTC-3′) were used to specifically amplify only mRNA generated from the knocked-in allele of the human Ush1c.216A gene, which is only present in correctly spliced mRNA (28 (link)). Mouse Gapdh primers (5′-GTGAGGCCGGTGCTGAGTATG-3′) and (5′-GCCAAAGTTGTCATGGATGAC-3′) were used to detect and measure endogenous murine Gapdh mRNA. Products were separated on a 6% non-denaturing polyacrylamide gel and quantitated using a Typhoon 9400 phosphorimager (GE Healthcare). Ush1c values were divided by Gapdh values and represented graphically following normalization to the Ush1c / Gapdh ratio of one intraperitoneal sample.
3
Circle Amplification Assay for DNA Analysis
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C-circle amplification assays were performed essentially as described14 (link)31 (link). Each DNA sample (50 ng) was incubated with or without 7.5 U ϕ29 DNA polymerase (NEB) in a 20 μl reaction containing 9.25 μl of premix (0.2 mg/ml BSA, 0.1% Tween 20, 1 mM ATP, 1 mM dTTP, 1 mM dGTP, 1xϕ29 buffer) at 30 °C for 8 h. Following heat inactivation of ϕ29 at 65 °C for 20 min, samples were separated on a 0.6% agarose gel in 0.5xTris-borate-EDTA (TBE) at 1.75 V/cm for 12–16 h. Gels were dried at 50 °C and were hybridized with a 32P-end-labeled single strand (CCCTAA)3 probe under native conditions as described32 (link). Gels were exposed to PhosphorImager screens and scanned using a Typhoon 9400 PhosphorImager (GE Healthcare).
4
Quantifying Viral Protein Synthesis
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Vero cells were plated on 12-mm coverslips placed in a 16-well dish designated for the immunofluorescence portion of this experiment. Alternatively, Vero cells were plated on 60-mm dishes designated for metabolic labeling. All cells were infected with rVSV at an MOI of 5. At 4 hpi, either 10 µg ml−1 puromycin (Sigma) or 5 µM PPMO designed to inhibit the translation of individual viral proteins was added. In the subset of cells designated for metabolic labeling, DMEM lacking l -methionine and l -cysteine (Life Technologies, Inc.) was supplemented with 17.5 µCi of [35S]-EasyTag express (PerkinElmer). At 7 hpi, cells were either fixed using 2% paraformaldehyde and prepared for immunofluorescence microscopy, or cells were lysed using lysis buffer (1% Nonidet P-40, 66 mM EDTA, 10 mM Tris-HCl, pH 7.4) and lysates analyzed by SDS-PAGE. Total cytoplasmic proteins were resolved by 10% SDS-PAGE and detected by phosphorimage analysis using a Typhoon 9400 Phosphorimager (GE Healthcare, Waukesha, WI).
5
Transcription Termination Efficiency Assay
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Promoter dependent transcription was carried out by preincubating 3 μl of S100, 100 ng plasmid DNA containing Mser-tRNA genes with different test terminators (2T-8T) in transcription buffer (100 mM KCl, 20 mM HEPES pH 7.5, 7 mM MgCl2 and 3 mM DTT) at 25°C. After 30 min the NTPs were added at 0.5 mM ATP, CTP, UTP, and 25 μM GTP plus 5 μCi [α-32P]GTP (10 mCi/ml; 3000 Ci/mmol) in final volume of 25 μl. After another 30 min the reactions were quenched with 200 μl stop buffer (10 mM EDTA, 1% SDS, 25 μg/ml Proteinase K) and incubated for 10 min. Products were recovered by phenol-chloroform extraction, ethanol precipitation, resolved by 6% polyacrylamide-urea gel and visualized by a Typhoon 9400 phosphorImager (GE Healthcare). Image flies were analyzed using Multigauge Software (Fuji).
6
Transcription of G-core DNA Template
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A dsDNA construct containing a T7 promoter and a downstream G-core (5′-TT(GGGGA)3GGGGTT-3′) was prepared by overlap extension polymerase chain reaction. Transcription was carried out with 0.05-μM dsDNA at 37°C for 1 h in 20 μl of transcription buffer of 40-mM Tris-HCl (pH 8.0), 30% (w/v) Dimethyl sulfoxide (DMSO), 50-mM KCl, 2 U/μl of T7 RNA polymerase (Thermo Scientific, MA, USA), 8-mM MgCl, 10-mM Dithiothreitol (DTT), 2-mM spermidine, 2-mM adenosine triphosphate (ATP), Uridine triphosphate (UTP) and Guanosine monophosphate (GMP), 1-mM Guanosine triphosphate (GTP) or 7-deaza-GTP and 0.005 U/μl inorganic pyrophosphatase, (Thermo Scientific, MA, USA). An equal volume of buffer containing 30% DMSO, 50-mM KCl, 1 μM of competitive DNA and the indicated RNases (0.8-μg/μl RNase A, 0.4-U/μl RNase H) was added and the mixture was maintained at 37°C for 1 h to terminate the transcription and digest the RNA. DNA samples were then resolved at 4°C, 8 V/cm on a 10% polyacrylamide gel in 1× Tris-borate-ethylenediaminetetraacetic acid (EDTA) buffer that contained 75-mM KCl (13 (link),19 (link)) and DNA detected by the fluorescence of carboxyfluorescein (FAM) dye labeled at the 5′ end of the non-template strand using a Typhoon 9400 phosphor imager (GE Healthcare, PA, USA).
7
Quantifying Ush1c mRNA Splicing
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Inner ears were rapidly dissected and immediately frozen in liquid nitrogen. Tissue was stored at −80°C until shipment on dry ice from Oregon to Illinois. RNA was isolated using Trizol reagent and one microgram was reverse transcribed using GoScript Reverse Transcriptase (Promega, Fitchburg, WI, USA). One microliter of cDNA was used in PCR reactions with GoTaq Green (Promega) supplemented with primers and α-32P-dCTP. Reactions were heated at 95°C for 2 min, followed by 37 cycles of: 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, and a final extension at 72°C for 2 min. Primers specific for mouse exon 2 (5′-CTCATTGAAAATGACGCAGAGAAGG-3′ and mouse exon 5 (5′-TCTCACTTTGATGGACACGGTCTTC-3′) were used to amplify cDNA generated from both the wild-type mouse Ush1c allele and the Ush1c allele with the mutated human USH1C insertion (USH1C c.216G>A). Products were separated on a 6% non-denaturing polyacrylamide gel and quantitated using a Typhoon 9400 phosphorimager (GE Healthcare). The amount of correct Ush1c splicing was quantified and presented as the percent of total Ush1c mRNA that is correctly spliced [(correctly spliced products/(correctly spliced products + cryptically spliced products) × 100].
8
Rheb GTP Loading Quantification
9
Tumor Tissue Sectioning and Imaging
10
Quantitative Analysis of Spliced USH1C mRNA
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Cochlear tissue for RNA isolation was rapidly dissected following euthanasia and snap-frozen in liquid nitrogen. Tissue was stored at −80°C until shipment on dry ice. Frozen tissues were homogenized in TRIzol solution (Thermo Fisher Scientific) using a PowerGen 1000 homogenizer (Thermo Fisher Scientific). Total RNA was purified from TRIzol reagent following the manufacturer’s recommendations. RNA was quantitated and purity assessed (A260/A280) using a BioPhotometer (Eppendorf). RNA (1 μg) was reverse transcribed using oligo(dT) primer and GoScript reverse transcriptase (Promega) following the manufacturer’s recommendations. Semiquantitative PCR was performed using 1 μL of cDNA and GoTaq Green (Promega) supplemented with primers and α-32P- deoxycytidine triphosphate (dCTP). Primers specific for human USH1C exon 3 (5′-GAATATGATCAGCTGACC-3′) and mouse exon 5 (5′-TCTCACTTTGATGGACACGGTCTTC-3′) were used to specifically amplify only mRNA generated from the knocked-in allele of the human USH1C c.216A gene, which is only present in correctly spliced mRNA. Mouse Gapdh primers (5′-GTGAGGCCGGTGCTGAGTATG-3′ and 5′-GCCAAAGTTGTCATGGATGAC-3′) were used to detect and measure endogenous murine Gapdh mRNA. Products were separated on a 6% non-denaturing polyacrylamide gel and quantitated using a Typhoon 9400 phosphorimager (GE Healthcare).
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