Nuclei from 4 × 107 cells were collected as described (Ramirez-Carrozzi et al. 2006 (link)). Nuclear pellets were sonicated with a Misonix 3000 sonicator and subsequently incubated overnight with ChIP-grade antibodies for Oct4 (R&D AF1759), Sox2 (R&D AF2018), H3K27ac (Active Motif 39133), H3K27me3 (Active Motif 39155), or H3K9me3 (Abcam ab8898). Antibody-bound complexes were collected using protein G Dynabeads (Invitrogen 10004D) and then reverse-cross-linked with proteinase K (Thermo Fisher Scientific EO0491) overnight at 60°C. Immunoprecipitated DNA was purified using phenol-chloroform extraction (Sigma P3803) and quantified by Qubit (Thermo Fisher Q32854). The enrichment of chromatin fragments was measured by qPCR with primer pairs designed to generate 100- to 125-bp amplified products within ±200 bp of the genomic sites of interest. Quantification of fold enrichment was calculated based on the fold change of the percentage of input between target genomic loci and a negative control region (Hbb-b2).
Phenol chloroform extraction
Manufactured by Merck Group
Sourced in United States
Phenol-chloroform extraction is a laboratory technique used to purify and isolate nucleic acids, such as DNA and RNA, from biological samples. It is a widely used method in molecular biology and genetics research. The process involves the use of organic solvents, such as phenol and chloroform, to separate the nucleic acids from other cellular components like proteins and lipids. The resulting aqueous phase contains the purified nucleic acids, which can then be further processed and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Rna storage solution, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Af1759, by R&D Systems (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Cleancap ag, by TriLink (1 mentions)
Hiscribe t7 kit, by New England Biolabs (1 mentions)
Nanodrop 8000 spectrophotometer, by Agilent Technologies (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Pulse field gel electrophoresis, by Bio-Rad (1 mentions)
Phire animal tissue direct pcr kit, by Thermo Fisher Scientific (1 mentions)
Nucleobond bac 100, by Takara Bio (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Proteinase k, by Bio-Rad (1 mentions)
Kod polymerase, by Merck Group (1 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (1 mentions)
Transit mrna transfection kit, by Mirus Bio (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
10 protocols using phenol chloroform extraction
1
ChIP-seq Analysis of Histone Modifications
2
Efficient mRNA Production for Base Editors
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Base editor mRNA was prepared by T7 run-off in vitro transcription using a custom plasmid template encoding for a T7 promoter, a minimal 5′-UTR, the base editor reading frame, 2× HBB 3′-UTR and a 110–120 bp poly(A) sequence54 (link). The plasmid template was linearized by BbsI-HF restriction digestion (NEB, R3539) and purified by phenol–chloroform extraction (Sigma-Aldrich, P2069). mRNA IVT was performed using the NEB HiScribe T7 kit (E2040S) and co-transcriptionally capped with ARCA (3′-O-Me-m7G(5′)ppp(5′)G RNA cap analogue, NEB, S1411L) or CleanCap AG (Trilink, N-7113). Partial (75–85%) or total UTP substitution with N1-methyl-pseudo-UTP (Trilink, N-1103) or 5-methoxy-UTP (Trilink, N-1093) was performed as indicated. Dephosphorylation with QuickCIP (NEB, M0525L) or DNase treatment (NEB, M0303L) was added after IVT reaction (30 min at 37 °C). IVT mRNA was purified using the NEB Monarch RNA Clean up kit (T2050L) or sparQ PureMag magnetic beads (MagBio, 95196) and resuspended in RNase-free water. mRNA was quantified using the Nanodrop-8000 spectrophotometer and quality control was performed using the Agilent Fragment Analyzer with RNA Kit-15NT (Agilent, DNF-471).
3
Isolation and Characterization of Liver RNA
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Frozen liver samples (~100 mg) were transferred to 1.3 mL of TRIzol (Life Technologies, Carlsbad, CA) and homogenized using a Mixer Mill 300 tissue homogenizer (Retsch, Germany). Total RNA was isolated according to manufacturer’s protocol with an additional phenol:chloroform extraction (Sigma-Aldrich, St. Louis, MO). Isolated RNA was resuspended in RNA storage solution (Life Technologies). Total RNA was quantified and assessed for purity by nanodrop (Thermo Scientific, Waltham, MA), Qubit (Life Technologies), and Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total RNA quality was also assessed using the A260/A280 ratio and by visual inspection on a denaturing gel. All sample processing and analysis was performed blinded to treatment group when possible.
4
Isolation of High-Quality Total RNA
5
Quantifying Gene Expression in ESCs, EpiLCs and PGCLCs
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Total RNA was isolated from ESCs, EpiLCs and PGCLCs (two biological replicates each, corresponding to two different clones, with further two technical replicates each) using phenol‐chloroform extraction (Sigma Aldrich, P2069) followed by ethanol precipitation and quantified by Nanodrop. cDNA was produced with a High‐Capacity RNA‐to‐cDNA kit (Thermo Fisher Scientific, 4387406) and was used for qRT–PCR analysis in triplicate reactions with Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, 4367659). The gene expression levels are presented as ∆∆Ct normalized with the mean Ct values of one housekeeping gene, Arbp, in a normalization sample (ESCs). The primer sequences used in this study are listed in Table 1 .
6
Generating Transgenic Mouse Lines
7
Comprehensive DNA Extraction from Water and Amphipods
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Total DNA from the water samples and the glass beads was extracted from the Sterivex filters as described in Neufeld et al. [45 (link)], where the filters were washed in a SET buffer and lysozyme solution, and the samples digested with proteinase K (all reagents from Sigma-Aldrich, St. Louis, MO, USA). The DNA was then rinsed by standard phenol-chloroform extraction (all reagents from Sigma-Aldrich, St. Louis, MO, USA) using a rotating hybridization oven (Hybridiser HB-1D, Techne, Bibby Scientific Limited, Staffordshire, UK). The DNA from the amphipods was extracted directly from whole animals in 6% Chelex 100, (Bio-Rad Laboratories Inc, Hercules, CA, USA) including proteinase K [3 (link)].
8
Rapid DNA Sequencing from Muscle Tissue
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DNA was extracted from a single GRZ male individual’s muscle tissue by grinding in liquid nitrogen followed by phenol-chloroform extraction (Sigma). The rapid sequencing kit (SQK-RAD004) and the ligation kit (SQK-LSK108) were sued to prepare six libraries and were sequenced on 6 MinION flow cells (R9.4.1). These runs yielded a total of 3.3 Gb of sequences after trimming and correction by HALC (Bao and Lan, 2017 (link)). For correction, Allpath-LG contigs (see below) and short reads from the 10X genomic run were used.
9
Generating Infectious RNA Clones
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Point mutations were introduced to pR16.11 with the Q5 site-directed mutagenesis kit (E0554S; New England BioLabs, Ipswich, MA) or the KOD polymerase (71085; Sigma-Aldrich, St. Louis, MO). Infectious clones were in vitro transcribed with the HiScribe T7 high-yield RNA synthesis kit (E2040S; New England BioLabs). RNA was collected by phenol-chloroform extraction (77617; Sigma-Aldrich) and transfected into HeLa-Ohio cells with the TransIT-mRNA transfection kit (MIR 2225; Mirus Bio, Madison, WI) according to the manufacturer’s conditions. Cells were harvested after 72 h and subjected to 2 freeze-thaw cycles, and the lysates were cleared by centrifugation at 10,000 × g at RT.
10
Total RNA Extraction from Buffy Coats and Fecal Samples
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Total RNA was extracted from buffy coats by phenolchloroform extraction (Sigma-Aldrich), and eluted with 50 μl DNAse/RNAse-free water. Total nucleic acids were extracted from fecal samples by glass fiber matrix binding and elution with the High-Pure PCR Template Preparation Kit (Roche) in 40 µl elution buffer.
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