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18 protocols using phenol chloroform isoamyl alcohol

1

Genomic DNA Extraction and PCR Genotyping

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Genomic DNA was purified with phenol/chloroform/isoamyl alcohol (Nakalai Tesque) from cells or mouse organs. Genotype PCR was performed with primers listed in Supplementary Table 1 using Ex-Taq polymerase (TaKaRa). Amplified DNA fragments were electrophoresed on 2% agarose gels containing ethidium bromide (Nakalai Tesque) and detected using an E-BOX imaging system with ultra-violet light (VILVER).
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2

DNA Extraction from Seaweed Samples

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Samples (G. chorda) were collected on the coast of Hakodate, Hokkaido Prefecture, Japan. Hexadecyltrimethylammonium Bromide (CTAB), tris-[hydroxymethyl]amino-methane (Tris), ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt, dihydrate (EDTA), Proteinase K (EC 3.4.21.64, from Tritirachium album), RNase A (EC 3.1.27.5, from bovine pancreas), porcine stomach pepsin (EC 3.4.23.1) and bovine pancreatic trypsin (EC 3.1.21.4) were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Phenol-chloroform-isoamyl alcohol (25:24:1) was purchased from NACALAI TESQUE, INC (Kyoto, Japan). Ala-Pro-p-nitroanilide (Ala-Pro-pNA) were also obtained from Bachem AG (Bubendorf, Switzerland). All other regents were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan).
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3

DNA Purification from Enzyme-Treated Mixtures

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The enzymes in the mixtures were inactivated by adding the same volume of phenol-chloroform-isoamyl alcohol = 25:24:1 (Nakalai Tesque) and mixing. The resulting emulsions were integrated into one tube, followed by the usual phenol-chloroform-isoamyl alcohol extraction and subsequent ethanol precipitation. The supernatant was transferred to a new tube and subjected to phenol-chloroform-isoamyl alcohol. The supernatant was then transferred to another new tube, and mixed with 500 μl of 1-butanol and centrifuged at 20,000 × g for 20 s. After the upper butanol phase was removed, fresh butanol was added and the solution was further mixed. This dehydration was repeated until the water phase fell below 450 μl. The DNA was precipitated by the addition of 2.5 volumes of ethanol after 50 μl of potassium acetate (300 mM, pH 4.8) was mixed with the water phase. The precipitated DNA was rinsed with 70% ethanol, dried, and dissolved in 20 μl of TE.
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4

Mitochondrial DNA Mutation Analysis

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Polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) (15 (link),70 (link)) was performed to measure the 3242A > G mutation rate of mtDNAs in myoblasts. Cultured cells were lysed in lysis buffer [20 mM Tris–HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA–NaOH (pH 8.0), and 0.1% SDS], followed by boiling at 60°C for 5 min and at 98°C for 2 min, and genomic DNA was extracted by phenol/chloroform/isoamyl alcohol (25:24:1) (Nacalai Tesque). The region including position 3243 of mtDNA was PCR-amplified with a set of primers (Supplementary Table S1). The PCR product was digested by the ApaI restriction enzyme and subjected to 10% native polyacrylamide gel electrophoresis (PAGE). Since 3243A > G mutation generates the ApaI site, the amplicon (666 bp) derived from mtDNA with the 3243A > G mutation was cleaved by ApaI into two fragments (234 and 432 bp). The gel was stained with SYBR Gold (S11494, Thermo Fisher Scientific), and the intensity of each band was quantified. The proportion of digested and undigested bands indicates the mutation rate of mtDNA with 3243A > G.
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5

In vitro Reconstitution of m1A947 in Human Mitochondrial 16S rRNA

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In vitro reconstitution of m1A947 was carried out essentially as described previously [24 (link)].
The 114-mer RNA segment including Helix 71 (G866-U979) of human mitochondrial 16S rRNA was transcribed in vitro using T7 RNA polymerase. Template DNA with T7 class III promoter for the 114-mer RNA segment was prepared by assembling the following DNA sequences:
5’-gctaatacgactcactataggcaccgcctgcccagtgacacatgtttaacggc-3’,
5’-gtgacacatgtttaacggccgcggtaccctaaccgtgcaaaggtagcataatcac-3’,
5’-gtgcaaaggtagcataatcacttgttccttaaatagggacctgtatgaatggctccacgagggtt-3’,
and 5’-aaccctcgtggagccattc-3’. In vitro transcription by T7 RNA polymerase was performed as described [40 (link)]. The transcript was purified by denaturing PAGE and quantified by measuring the optical density at 260 nm. The reaction mix (50 μL), consisting of 25 mM Hepes-KOH (7.5), 100 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 1 mM Ado-Met, 1.5 μg total RNA, and 1 μM His-tagged TRMT61B, was incubated for 2 h at 37°C, followed by adding phenol-chloroform isoamylalcohol (Nacalai) to terminate the reaction. Total RNA was recovered by ethanol precipitation and subjected to primer extension as described above.
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6

In Vitro Reconstitution of tRNA t6A37

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In vitro reconstitution of t6A37 on tRNA was carried out at 37 °C for 1 h in a 20-μL reaction mixture containing 100 mM Tricine-NaOH (pH 8.0), 2 mM DTT, 15 mM MgCl2, 1 mM ATP, 5 mM L-Thr, 40 mM NaHCO3, 1 μM mt-tRNA, 1 μM YRDC (17–279a.a.), and 2 μM OSGEPL1 (35–414a.a.). The mt-tRNAs were extracted with phenol/chloroform/isoamyl alcohol (25:24:1, pH 5.2) (Nacalai Tesque), dialyzed, digested with RNase T1, and subjected to LC/MS analyses to detect and quantify t6A37 formation.
For kinetic analyses, initial velocity of t6A37 formation was measured at 37 °C in a 10-μL reaction mixture (for each data point) containing 100 mM Tricine-NaOH (pH 8.0), 2 mM DTT, 15 mM MgCl2, 18.75–150 μM ATP, 14.275–114.2 μM [14C] L-Thr, 5–40 mM NaHCO3, 0.4–3.2 μM mt-tRNA, 1 μM YRDC (17–279a.a.), and 2 μM OSGEPL1 (35–414a.a.). To stop the reaction, the mixture was spotted on a Whatman 3MM filter paper, soaked in 5% TCA, washed three times with 5% TCA, and rinsed with ice-cold ethanol. Radioactivity of [14C] L-Thr on the dried paper was measured by liquid scintillation counting on a Tri-Carb 2810TR (PerkinElmer).
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7

Genotyping mRFP1 and BAT Hmgcr KO Mice

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mRFP1-positive mice and BAT-specific Hmgcr KO mice were identified by genotyping, as described previously.58 (link) Briefly, the tail (2–3 mm) of mice or tissues were incubated with a lysis buffer (10 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.5% SDS, and 100 mM NaCl) containing 1 mg/mL proteinase K (Nacalai Tesque) at 50°C overnight. After the samples were dissolved, 200 μL of phenol:chloroform:isoamyl alcohol (25:24:1; Nacalai Tesque) was added, and centrifuged at 13,800 ×g for 5 min at 4°C. The supernatant was transferred to a new tube, and 200 μL of chloroform was added. After centrifugation at 14,000 ×g for 5 min at 4°C, 160 μL of the supernatant was transferred to new tube containing 16 μL of 3 M Na-acetate and 400 μL of ethanol and centrifuged at 19,000 ×g for 20 min at 4°C. The DNA pellet was dissolved in TE buffer (pH 8.0). Genomic DNA was amplified using EmeraldAmp MAX PCR Master Mix (Takara) in a thermal cycler and confirmed by agarose gel electrophoresis. Primer sequences used are listed in Table S2.
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8

Genomic DNA Extraction from Fish Fins

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Fish were anesthetized in 0.004% tricaine (ethyl 3-aminobenzoate methanesulfonate, MS-222, Sigma-Aldrich, St. Louis, MO, USA) and subjected to fin clip for a length of 2 mm. The fins were homogenized using Biomasher II (Nippi, Tokyo, Japan) in 290 μL of Genome Lysis Buffer (10 mM Tris–Cl (pH 8.0), 10 mM EDTA, 150 mM NaCl, 0.1% SDS, 33 ng/mL Proteinase K) for 3 h at 56 °C with thorough mixing by gentle inversion. Equal amounts (300 μL) of phenol/chloroform/isoamyl alcohol (25:24:1, pH 7.9) (Nacalai Tesque, Kyoto, Japan) were used to purify genomic DNA. After isopropanol (Nacalai Tesque) precipitation, the genome was washed with 80% EtOH followed by resuspension in 5 mM Tris–HCl (pH 8.0). Further clean-up of genomic DNA was conducted using AMpure XP beads (Beckman Coulter, Brea, CA, USA) according to the manufacturer’s manual. The concentration of the genomic DNA was measured using Quantus Fluorometer (Promega, Madison, WI, USA) and adjusted to 20 ng/μL.
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9

DNA Extraction from Mucosal Swabs

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An overview of this method is presented in Figure 1C. The swab samples in the SNET buffer, including mucosae, were incubated at 56 °C for 30 min to disassemble the protein, followed by vigorous shaking. Subsequently, the SNET buffer, including mucosae DNA, was eluted from the swab samples by centrifugation at 5000× g for 1 min at room temperature, via the use of a cell strainer. The DNA pellets were obtained from 800 µL of the eluted solutions by applying equal volumes of phenol/chloroform/isoamyl alcohol (Cat# 25970-56; Nacalai Tesque, Nakagyo, Kyoto, Japan) with a general centrifugation and precipitation method. The pellets were then dissolved in 50 µL of Milli-Q water containing Ribonuclease A (10 µg/mL) and incubated at 37 °C for 10 min to degrade residual RNA. The solutions were then purified using NucleoSpin Gel and a PCR clean-up kit (Cat# U0609B; Takara Bio, Kusatsu, Shiga, Japan), the latter of which was used according to the manufacturer’s instructions; the final elution volume was 30 µL. The purified DNA solutions were subjected to gel electrophoresis to confirm the degree of degradation with a positive control of intact DNA that was extracted from adipose-derived mesenchymal stem cells, called the 1117 cell (our original), of a Toy Poodle.
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10

Chromatin Immunoprecipitation in Yeast

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Log-phase yeast cultures were fixed in 3% paraformaldehyde (Sigma-Aldrich) for 30 min on ice. The reaction was quenched with the addition of 0.0625 (v/v) of 2.5 M glycine (Thermo Fisher Scientific, Wilmington, DE, USA) and the samples washed twice in PBS, sonicated, and lysed. The samples were then immunoprecipitated with specific antibodies, which were later bound to protein A agarose (Life Technologies, Carlsbad, CA) and protein G agarose (Roche Diagnostic, Indianapolis, IN) (1:1). The agarose was washed and reverse crosslinked by incubating for >8 h at 65°C. The immunoprecipitants were then digested with 20 mg/ml Proteinase K (Roche Diagnostics, Basel, Switzerland) for 2 h at 37°C and extracted with 1 vol phenol:chloroform:isoamyl alcohol (25:24:1) (Nacalai Tesque, Kyoto, Japan). The aqueous layer was then ethanol precipitated, dried, and analyzed with PCR using centromere-specific primers (see Supplementary Table S2). The previously published sonication method and buffer composition were closely followed (30 (link),41 (link)).
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