Phenol chloroform isoamyl alcohol

Manufactured by Thermo Fisher Scientific

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Phenol/chloroform/isoamyl alcohol is a mixture commonly used in the extraction and purification of nucleic acids, such as DNA and RNA, from biological samples. It is a liquid solution that acts as a reagent in the process of isolating and separating these genetic materials from other cellular components.

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Chloroform (747 citations) Phenol (68 citations) Phenol/chloroform (56 citations) Ultra-Pure phenol:chloroform:isoamyl alcohol (25 citations) Acid phenol:chloroform:isoamyl alcohol (7 citations) Isoamyl alcohol (6 citations) Neutral phenol–chloroform-isoamyl alcohol (4 citations) Phenol:chloroform:isoamyl (3 citations) UltraPure™ Phenol:Chloroform:Isoamyl Alcohol reagent (2 citations) Phenol-chloroform-isoamyl alcohol mix (2 citations)

121 protocols using phenol chloroform isoamyl alcohol

DNA Extraction from i6A-Treated Cells

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After incubating cells in medium containing 0, 1, or 2 µM i⁶A for 24 or 48 h, cells were rinsed with PBS three times, trypsinized, and collected. The cell pellets were lysed in 1 ml of Proteinase K buffer (20 mM Tris-HCl [pH 8.0], 5 mM EDTA, 400 mM NaCl, 0.3% sodium dodecyl sulfate) and sonicated for a total of 24 sec using Sonifier 250 (Branson) at power 4. Then, 10 µL of Proteinase K (NEB) was added to the solution, and the samples were incubated at 55°C for 1 h for protein digestion, followed by Proteinase K inactivation using phenol-chloroform-isoamylalcohol (Invitrogen). Nucleic acids were collected by isopropanol precipitation, cleaned by 75% ethanol washing, and eluted into 10 mM Tris-HCl (pH 8.0)/1 mM EDTA (pH 8.0). RNA in the solution was digested using RNaseA (Qiagen), and RNaseA was inactivated using phenol-chloroform-isoamylalcohol (Invitrogen). The remaining DNA was isopropanol-precipitated, 75% ethanol-washed, and dissolved into MilliQ water.

Yakita M., Chujo T., Wei F.Y., Hirayama M., Kato K., Takahashi N., Naganuma K., Nagata M., Kawahara K., Nakayama H, & Tomizawa K. (2022). Extracellular N6-isopentenyladenosine (i6A) addition induces cotranscriptional i6A incorporation into ribosomal RNAs. RNA, 28(7), 1013-1027.

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Ribosome Profiling with Puromycin Treatment

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HEK293T cells were grown to ∼60% confluency and then treated with 100 μM s⁴U or s⁶G for 4 h. Plates were washed in ice-cold PBS and each plate was scraped into its own LoBind tube. Control samples were lysed in cycloheximide lysis buffer (20 mM Tris–HCl pH 7.5, 10 mM MgCl₂, 200 mM KCl, 1% Triton, 0.2 mg/ml cycloheximide, 4 mM EDTA) and passed 10× through a 26-gauge needle. Lysate was cleared at 20 000 × g for 10 min at 4°C. Puromycin treated samples were resuspended in Puromycin lysis buffer (20 mM Tris–HCl pH 7.5, 5 mM MgCl₂, 200 mM KCl, 1% Triton, 4 mM EDTA), passed 10× through a 26-gauge needle, and cleared at 20 000 × g for 10 min at 4°C. Puromycin (VWR) was added to 2 mM, and samples were incubated on ice for 20 min, and then at 36°C for 20 min. MgCl₂ was added up to 10 mM (23 (link)). After Puromycin treatment, samples were mixed.
Following ultracentrifugation, fractions were collected into phenol:chloroform:isoamyl alcohol (Fisher Scientific). In total, one phenol extraction was performed, with two additional chloroform extractions. RNA was ethanol precipitated, and DNA removed with TurboDNase.

Courvan M.C., Niederer R.O., Vock I.W., Kiefer L., Gilbert W.V, & Simon M.D. (2022). Internally controlled RNA sequencing comparisons using nucleoside recoding chemistry. Nucleic Acids Research, 50(19), e110.

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Bacterial Transcriptome Profiling from Infected Lungs

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In vitro and infected mouse lung tissue samples were thawed and nucleic acid was isolated by organic partition. Samples were treated with DNAse (Fermentas, Burlington, Ontario) for 30 minutes and purified by phenol/chloroform/isoamyl alcohol (25:24:1) (Fisher Scientific, Pittsburgh, PA) extraction and ammonium acetate precipitation. Biological replicates (not pooled) were submitted to the CSU Next Generation Sequencing core for sample processing and sequencing. Briefly, RNA sample quality was determined on an Agilent 2100 Bioanylizer and samples with a RIN value greater than 8 passed the criteria for sequencing. Host transcripts were removed using MICROBEnrich (Life Technologies, Carlsbad, CA), sample libraries were prepared using the Ion Total RNA-Seq kit v2 (Life Technologies), and multiplexed on a P1 chip using Ionxpress RNA-Seq 1–16 kit (Life Technologies). Whole bacterial transcriptome sequencing was performed using the Ion Proton Next Generation Sequencer (Life Technologies).

Cummings J.E, & Slayden R.A. (2017). Transient In Vivo Resistance Mechanisms of Burkholderia pseudomallei to Ceftazidime and Molecular Markers for Monitoring Treatment Response. PLoS Neglected Tropical Diseases, 11(1), e0005209.

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Extraction and Quantification of Total RNA

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Samples of 700 μl were withdrawn directly from cultures and collected into 2 ml tubes containing 100 μl of 8× lysis buffer (92 μl dH₂O, 160 μl 3M NaOAc, 1200 μl 10% SDS, 48 μl 0.5 M EDTA) and 800 μl of 5:1 phenol:chloroform (pH 4.1, Fisher Scientific) pre-warmed to 65°C. Samples were processed at 65°C for five min with intermittent shaking, then centrifuged at 4°C. The aqueous layer was transferred to a new Eppendorf tube and extracted once or twice with 25:24:1 phenol:chloroform:isoamyl alcohol (pH 6.7, Fisher Scientific) or with pure chloroform (Fisher Scientific). Total RNA was alcohol-precipitated from the final aqueous fraction and suspended in Tris-EDTA (TE) buffer or DEPC-treated water. The final RNA concentration was quantified using a Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific).

Cameron T.A., Matz L.M., Sinha D, & De Lay N.R. (2019). Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments. Nucleic Acids Research, 47(16), 8821-8837.

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Intestinal Bacterial Detection by qPCR

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DNA was extracted from an intestinal homogenate and used as template for model strain detection by quantitative PCR. 500 µl of intestinal homogenate was mixed with 210 µl of 20% SDS, 500 µl phenol:chloroform:isoamyl-alcohol (24:24:1, Fisher Scientific), and 500 µl of 0.1-mm zirconia beads (BioSpec, Bartlesville, OK, USA). The mixture was then lysed with a bead beater at 2,400 RPM for 3 min. The qPCR assay to detect model strains was performed using a universal 16S rRNA gene primer set to detect total bacterial load (forward primer: 5′-GTGSTGCAYGGYTGTCGTCA-3′, reverse primer: 5′-ACGTCRTCCMCACCTTCCTC-3′) (Horz et al., 2005 (link)). Each reaction was done in triplicates with 12.5 µl of iQ SYBR Green Supermix (BIO-RAD2), 1 µl of 10µM forward and reverse primers, 5.5 µl nuclease-free water, and 5 µl of extracted DNA (200 ng/µl).

Cho J.Y., Liu R, & Hsiao A. (2022). Microbiota-Associated Biofilm Regulation Leads to Vibrio cholerae Resistance Against Intestinal Environmental Stress. Frontiers in Cellular and Infection Microbiology, 12, 861677.

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DNA Extraction from Swab Samples

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DNA was extracted from samples using a standard phenol:chloroform method [39 ]. Briefly, the cotton tip was removed from a swab and incubated overnight at 56 °C in 400 μl DNA extraction buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml Proteinase K). The swab was removed to a Spin-X filter (Corning, Tewksbury, MA) and the tube centrifuged. Four hundred microliters of 25:24:1 phenol/chloroform/isoamyl alcohol (Fisher, Scientific, Norcross, GA), were added, and the phases were separated in a Phase Lock Gel Tube (2 ml, heavy, Eppendorf, Boulder, CO) according to the manufacturer's protocol. DNA was precipitated for at least 1 h in 1 ml (2.5 vol) absolute ethanol at −20 °C and pelleted by centrifugation. The pellet was washed twice with 1 ml (2.5 vol) 70% ethanol and dried in a 56 °C incubator (∼10–20 min). The DNA was re-solubilized in 30 μl sterile water by overnight incubation in a 56 °C water bath (12–18 h).

Tang J., Ostrander J., Wickenheiser R, & Hall A. (2019). Touch DNA in forensic science: The use of laboratory-created eccrine fingerprints to quantify DNA loss. Forensic Science International, 2, 1-16.

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RISC-IP Assay of miR-210 Regulation

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RISC-IP assay was performed as previously described (Hu et al., 2021 (link)). Cultured primary uterine arterial smooth muscle cells (passages 2–3) were transfected with 100 nmol/L miR-210 mimic or the negative scrambled control (Qiagen) by using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. Cells were harvested 24 hours after transfection and washed in ice-cold PBS followed by complete lysis buffer (Active Motif) at 4 °C for 10 minutes. RISC-IP of the lysate was conducted using the miRNA Target IP Kit (Active Motif) according to the manufacturer’s instructions. The RNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, Fisher Scientific) once, chloroform once and precipitated and resuspended in RNase-free water. The precipitated RNA was subjected to RT-qPCR using primers specific for the sheep ISCU 3′-UTR. β-actin was used as an internal control. The relative abundance of ISCU transcript pulled down by Ago1/2/3 antibody was calculated by the 2^−ΔΔCT method and is presented as the fold induction relative to the control. Primers used were 5’-GTTTCCTGCAGCTCACTTGC (forward) and 5’-CCCAAACCTGAGACTTCGCT (reverse) for ISCU and 5′-GCAGGTCATCACCATCGGCAAT (forward) and 5’-ACCGTGTTGGCGTAGAGGTCCT (reverse) for β-actin.

Hu X., Song R., Dasgupta C., Romero M., Juarez R., Hanson J., Blood A.B., Wilson S.M, & Zhang L. (2022). MicroRNA‐210‐mediated mtROS confer hypoxia‐induced suppression of STOCs in ovine uterine arteries. British Journal of Pharmacology, 179(19), 4640-4654.

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Phenol-Chloroform DNA Extraction Protocol

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Each tube containing bacterial culture pellet or tissue lysate in 500 μL of TE was subjected to DNA extraction by a protocol that utilizes phenol/chloroform/isoamyl alcohol as previously described [8 (link)]. Briefly, tubes were incubated in a heat block for 30 min at 100 °C and then placed on ice for 15 min. Tubes were then centrifuged at 12,000 RPMs at 4 °C for 10 min. Supernatants were transferred into 2.0 mL Phase Lock Gel™ tubes (Fisher Scientific^®) and then mixed with 200 μL of phenol/chloroform/isoamyl-alcohol (Fisher Scientific^®). Tubes were centrifuged at 12,000 RPMs at 4 °C for 5 min, where supernatants transferred into new 1.5 mL microcentrifuge tubes containing 100% chilled ethanol and stored at − 20 °C overnight. Next day, tubes are thawed and centrifuged at 12,000 RPMs at 4 °C for 10 min and the supernatants discarded. DNA pellets were washed with 80% chilled ethanol, dried in a speedvac for 15 min and re-suspended in 50 μL TE buffer for storage at − 20 °C until further use.

Sharp R.C., Naser E.S., Alcedo K.P., Qasem A., Abdelli L.S, & Naser S.A. (2018). Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease. Gut Pathogens, 10, 51.

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Genomic DNA Extraction and Sonication

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10 – 30 mL of S. aureus or E. coli or culture grown to saturation were pelleted and washed with 1 volume of TE buffer (pH 8.0). Pellets were re-suspended in ~3 mL of ice-cold TE buffer (pH 8.0). Every 500 μL of re-suspended cells was mixed with 500 μL of ice-cold Phenol/Chloroform/Isoamyl alcohol (25:24:1) (Fisher Scientific). The mixture was transferred into a 2 mL microtubes pre-filled with ~0.25 cm³ of glass beads (0.1 mm) on ice. Cells were disrupted using FastPrep-24 5G™ Homogenizer (MPBio) at 4°C. The default S. aureus or E. coli setting was used. The homogenized mixture was centrifuged at 16,000 rcf. for 10 min at room temperature. The aqueous phase was collected and mixed with 500 μL of chloroform and centrifuged as above. The aqueous phase was collected again, mixed with 1 mL of isopropanol, gently inverted several times, incubated for 10 min at room temperature and centrifuged. Precipitated genomic DNA was washed with 1 mL of 75% ethanol, air-dried and dissolved in 50–300 μL of water.
Genomic DNA was sonicated in 130 μL total volume in microTUBE AFA Fiber Pre-Slit Snap-Cap 6 × 16 mm tubes (Covaris) using the Covaris S220 Focused-ultrasonicator to a fragment size of 150 bp. The sonicated DNA was dialysed before electroporation.

Jiang W., Oikonomou P, & Tavazoie S. (2020). Comprehensive Genome-wide Perturbations via CRISPR Adaptation Reveal Complex Genetics of Antibiotic Sensitivity. Cell, 180(5), 1002-1017.e31.

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Extraction of Genomic DNA from Archived Breast Tissues

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Genomic DNA was extracted from archived breast cancers and matching benign epithelial tissues following a previously published method [19 (link), 27 (link)]. In brief, for FFPE samples, a 10-μM thick tissue section was deparaffinized in 1 mL of octane (Fisher Scientific, Suwanee, GA). The tissue pellet was re-suspended in 180 μL of digestion buffer [(50 mM Tris, pH 8; 1 mM EDTA; 1% Tween 20) plus 20 μL of proteinase K (20 mg/ml) (Fisher Scientific)], and the mixture was incubated for 24 hr at 56°C. Samples were heated to 95°C, then 200 μL of phenol/chloroform/isoamylalcohol (Fisher Scientific) (25:24:1, pH 6.7) was added. The aqueous layer was transferred into Microcon YM-100 filter tubes (Fisher Scientific, USA). The DNA was eluted from the filter tubes by adding 125 μL of TE buffer (10 mM Tris hydrochloric acid; 0.1 mM EDTA, pH 8). DNA quality and concentration were determined by spectrophotometry. Genomic DNA from snap-frozen tissues was extracted by use of DNeasy Tissue Kits (Qiagen). The quality of DNA, defined by the ratio of E260nm/280nm = 1.8–2.0, was ascertained for all samples using NanoDrop (Fisher Scientific).

Putcha B.D., Jia X., Katkoori V.R., Salih C., Shanmugam C., Jadhav T., Bovell L.C., Behring M.P., Callens T., Messiaen L., Bae S., Grizzle W.E., Singh K.P, & Manne U. (2015). Clinical Implications of Rabphillin-3A-Like Gene Alterations in Breast Cancer. PLoS ONE, 10(6), e0129216.

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