E. coli MG1655 was obtained from the ATCC. MG1655 ΔnudC:Kan was previously reported (Bird et al. 2016 (link)). E. coli MG1655 or MG1655 ΔnudC:Kan cells were grown overnight and then back diluted 1:100 in 25 mL LB (10 g Bacto-tryptone, 5 g Bacto-yeast extract and 10 g NaCl per liter) containing 25 µg mL–1 kanamycin (only for the ΔnudC:Kan strain) in 125 mL flasks (Bellco). Cultures were shaken at 220 rpm at 37°C. When cell density reached an OD600 ∼ 3.4 (∼8 h), 5 mL of the cell suspension was centrifuged (1 min, 10,000g at room temperature) to collect cells, supernatants were removed, and cell pellets were rapidly frozen on dry ice. To extract total RNA, frozen pellets were resuspended in 1 ml of TRI Reagent solution (Molecular Research Center). Samples were incubated at 70°C for 10 min and then centrifuged (10 min, 21,000g, 4°C) to remove insoluble material. The supernatant was transferred to a fresh tube and 200 µL of chloroform was added, samples were mixed by vortexing, and then centrifuged (10 min, 21,000g, 4°C). The aqueous phase was transferred to a fresh tube, extracted with acid phenol:chloroform twice (Ambion), and RNA transcripts were recovered by ethanol precipitation, washed with 75% ethanol at 4°C and resuspended in RNase free water (Ambion).
Acid phenol chloroform
Manufactured by Thermo Fisher Scientific
Sourced in United States
Acid phenol/chloroform is a laboratory reagent used for the extraction and purification of nucleic acids, such as DNA and RNA, from biological samples. It is a mixture of phenol, chloroform, and isoamyl alcohol, which helps to separate the nucleic acids from other cellular components.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Tri reagent, by Merck Group (3 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Maxima h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Glycoblue, by Thermo Fisher Scientific (3 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Anti m3 g m7g cap, by Synaptic Systems (2 mentions)
Rnasin ribonuclease inhibitor, by Promega (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Rq1 dnase, by Promega (2 mentions)
Typhoon 5 imager, by Cytiva (2 mentions)
Hispur ni nta magnetic beads, by Thermo Fisher Scientific (2 mentions)
M280 sheep anti mouse igg dynabeads, by Thermo Fisher Scientific (2 mentions)
Pefabloc, by Roche (2 mentions)
Dna free kit, by Thermo Fisher Scientific (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Takara Bio (2 mentions)
49 protocols using acid phenol chloroform
1
Transcriptomic Analysis of E. coli MG1655
2
RNA Extraction from Cyanidioschyzon merolae
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C. merolae cultures (10 ml) were harvested after 1 h of growth in the selected media. Cell pellets were resuspended in 500 µL cold phenol RNA lysis buffer (0.5 M NaCl, 0.2 M TrisHCl (pH 7.5), 0.01 M EDTA, 1% SDS) and were sonicated briefly to shear the genomic DNA. Total RNA was extracted two times with acid phenol/chloroform (pH 4.5, Ambion), followed by chloroform extraction. RNA was EtOH precipitated and resuspended in dH2O and then treated with Turbo DNase (Ambion) following the manufacturer’s instructions.
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C. merolae cultures (10 ml) were harvested after 1 h of growth in the selected media. Cell pellets were resuspended in 500 µL cold phenol RNA lysis buffer (0.5 M NaCl, 0.2 M TrisHCl (pH 7.5), 0.01 M EDTA, 1% SDS) and were sonicated briefly to shear the genomic DNA. Total RNA was extracted two times with acid phenol/chloroform (pH 4.5, Ambion), followed by chloroform extraction. RNA was EtOH precipitated and resuspended in dH2O and then treated with Turbo DNase (Ambion) following the manufacturer’s instructions.
3
Efficient Total RNA Extraction from Liver
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Total RNA was extracted from fresh liver tissues, with 5 biological replicates per group. Time of tissue collection was standardized over a 4-hour period, with necropsies starting at 8 AM local time. The left lateral lobe was rapidly removed, rinsed with phosphate buffered saline, immediately homogenized in Trizol (Thermo Fisher Scientific) using a Kimble motorized pellet pestle, and phase separation was performed according to the manufacturer’s instructions. The Trizol aqueous phase was then purified using Acid Phenol:Chloroform (Ambion) according to the manufacturer’s instructions. An equal volume of 50% ethanol was added to the aqueous layer and the RNA was purified using the RNeasy Mini Kit (Qiagen) with RNase-Free DNase treatment (Qiagen), according to the manufacturer’s instructions. RNA integrity was assessed using the Agilent Bioanalyzer with the RNA 600 Pico Kit (Agilent). The RNA Integrity Number (RIN) for total RNA samples ranged from 9.3 to 9.7.
4
Cap-IP Analysis of RNA Transcripts
5
Sperm RNA Isolation using mirVana
6
Quantifying Transposon Expression in Drosophila
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Total RNA was extracted from ovaries of 1- to 2-day-old females, using TRI reagent (Sigma). Samples were then treated with Turbo DNase (Ambion) and the RNA was precipitated using acid phenol: chloroform (Ambion). cDNA was synthesized from 2 µg RNA using Maxima H Minus First Strand cDNA synthesis kit (Thermo Scientific). Random hexamers were used for reverse transcription, and to enrich rp49 cDNA a specific RT primer (5′-TTGGAGGAGACGCCG-3′) was added to the mixture. The resulting cDNA was used for RT-qPCR using HeT-A-1, 18S rRNA and pre-rp49 primers described by (Zhang et al., 2012 (link)). RT-qPCR was performed in a CFX96 Real-Time machine (Bio-Rad) using DyNAmo Flash SYBR Green (ThermoScientific). The expression level of HeT-A was quantified relative to 18S rRNA and pre-rp49. A minimum of three biological replicates were tested for each genotype. The graph shows the average and the error bars indicate the standard error of the mean (SEM).
7
Identification of shyB Transcription Start Site
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The shyB transcription start site was identified with 5′-rapid amplification of cDNA ends (5′-RACE). To obtain a shyB transcript, the Δzur mutant was grown in LB at 37°C until cells reached mid-log phase (optical density at 600 nm [OD600], 0.5), and RNA was extracted using TRIzol reagent and acid-phenol-chloroform (Ambion). DNA contamination was removed through two RQ1 DNase (Promega) treatments and additional acid-phenol-chloroform extractions. cDNA synthesis was performed with MultiScribe reverse transcriptase (Thermo Fisher) and a shyB-specific primer (SM270). cDNA was column purified and treated with terminal transferase (New England BioLabs) to add a homopolymeric cytosine tail to the 3′ end. The cDNA was amplified through two rounds of touchdown PCR with a second gene-specific primer (SM271) and the anchored abridged primer (Thermo Fisher). The PCR product was Sanger sequenced using primer SM271.
8
Cas6-mediated RNA cleavage assay
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Two micromolars purified recombinant Cas6 was incubated with 1–5 nM [γ-32P] ATP-labelled RNA (CD, AB or ncRNA60) at 60°C in nuclease reaction (NR) buffer (20 mM NaH2PO4/Na2HPO4 pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5 mM dithiothreitol). This buffer supported a higher Cas6 activity than that reported previously (14 (link)). To stop the reaction, aliquots of 10 μl were quenched by addition to 30 μl acid phenol/chloroform (Ambion), vortexed briefly and centrifuged at 15 000 × g for 1 min. Five microlitres of the upper aqueous phase was removed and mixed 1:1 with formamide. Samples were heated at 95°C for 2 min and loaded onto a pre-run 20% denaturing polyacrylamide gel (20% acrylamide, 8 M urea, 1× Tris-borate-EDTA (TBE)) then electrophoresed at 80 W for 90 min in 1× TBE running buffer. Following electrophoresis, gels were scanned by phosphorimaging and analysed using Fuji Imagegauge software as described previously (24 (link)).
9
SARS-CoV-2 N-gene RNA binding assay
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For the experiments shown in Fig. 1b and Supplementary Fig. 1b, c , 32P-labeled RNA (25 nM) was incubated with TtCsmCsm3-D34A (160 nM) targeting SARS-CoV-2 N-gene in 1× Binding Buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP) for 20 min at 65 °C. The reaction mixtures were added to 10 µL of HisPur Ni-NTA Magnetic beads (ThermoFisher) equilibrated in Binding Buffer and incubated on ice 30 min with vortexing every 10 min. The beads were separated from the supernatant using a magnet and washed with 50 µL 1× binding buffer. The RNA was extracted from supernatant (unbound fraction) and beads (bound fraction) using Acid Phenol: chloroform (Ambion). Extracted RNA was resolved using UREA-PAGE, exposed to a phosphor screen, and imaged on a Typhoon 5 imager (Amersham). Bands corresponding to the IVT RNAs were quantified using ImageJ v1.52t and the percent bound calculated [bound/(bound + free)*100%].
10
Isolation and Characterization of Defective Interfering RNA
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Thirty-microgram of total RNA was treated with DNase Ⅰ (Takara Bio, Japan) and S1 nuclease (Takara Bio, Japan) and separated on 1% agarose gel by gel electrophoresis. Purifying defective interfering RNA from agarose gel was conducted by repeated cycles of freezing in liquid nitrogen and thawing in a 42 °C water bath. The aqueous phase was obtained with 1:1 (v:v) acid-phenol:chloroform (Ambion, USA). dsRNAs in the aqueous phase were recovered by ethanol precipitation and centrifugation. The obtained dsRNA pellet was suspended in DEPC-water. cDNA was synthesized using GoScriptTM reverse transcriptase (Promega, USA) with random hexamer or primer mixture, including 3′-UTR sequences of each FgV2 RNA. cDNA was amplified by PCR with a random or virus-specific primer set for the UTR of each FgV2 RNA. Primer sets are listed in Table S2. PCR product was cloned to pGEM-T easy vector (Promega, USA), and the sequence was analyzed by Macrogen (Seoul, South Korea). Sequences of defective interfering RNA were aligned with FgV2 RNAs using SeqMan software (DNASTAR, Madison, WI, USA)
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