The DNA samples from (KOR1 -miak/miak × B6J) F2 and (B6J-miak/miak congenic × B6J) F2 mice were typed for multiple microsatellite markers located throughout the mouse genome. The markers were selected from the Microsatellite Database of Japan (http://www.shigen.nig.ac.jp/mouse/mmdbj/top.jsp ) based on size variation between PCR products from B6J- and Japanese-derived strains (MSM/Ms and JF1Ms). The genotypes were then analyzed for cosegregation with the mutant phenotype, which is the easily identifiable characteristic of small eyes. The PCR conditions for genotyping were as previously described [20] (link). The polymorphisms of the PCR products were visualized on 4% agarose (3% Agarose XP and 1% Agarose S, Nippon gene, Tokyo, Japan) stained with ethidium bromide.
Agarose s
Manufactured by Nippon Gene
Sourced in Japan
Agarose S is a high-quality agarose powder used for gel electrophoresis. It is designed to provide consistent and reliable results in DNA and RNA separation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Abi prism 3100 genetic analyser, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Ex taq pcr kit, by Takara Bio (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Midori green xtra, by Nippon Genetics (1 mentions)
Fas 5 gel imaging system, by Nippon Genetics (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Dna ligation kit, by Takara Bio (1 mentions)
Kanamycin, by Fujifilm (1 mentions)
Lb agar, by Merck Group (1 mentions)
Mighty ta cloning kit, by Takara Bio (1 mentions)
Ampicillin, by Fujifilm (1 mentions)
Agarose 1, by Dojindo Laboratories (1 mentions)
Gelred, by Fujifilm (1 mentions)
Mighty mix, by Takara Bio (1 mentions)
Sybr safe, by Thermo Fisher Scientific (1 mentions)
Midori green direct, by Nippon Genetics (1 mentions)
Safelook load green, by Fujifilm (1 mentions)
Gelred, by Biotium (1 mentions)
12 protocols using agarose s
1
Genetic Mapping of Eye Mutation
2
Molecular Marker Amplification and Sequencing
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PCR amplifications were conducted using an Ex Taq PCR kit (TaKaRa, Kyoto, Japan) for three different molecular markers: cytochrome c oxidase subunit I (COI ), 16S rRNA PageBreak(16S ), and 18S rRNA (18S ). Two oligonucleotide primers (0.2 µM each) and <1 µg of DNA template were added to the reaction mixtures. Thermal cycling was performed as follows: denaturing at 96 °C for 20 s, annealing at 55 °C for 45 s, and extension at 72 °C for 2 min for a total of 35 cycles. The oligonucleotide primer sequences used for the PCR amplification are shown in Table 1 . The molecular sizes of the PCR products were confirmed with 1.2% Agarose S (Nippon Gene, Toyama, Japan) gel electrophoresis. The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). The DNA sequencing reaction was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Specific primers for each gene were used in sequencing reactions according to the manufacturer’s recommended procedure (Table 1 ). Sequencing was performed using an ABI PRISM 3100 genetic analyser (Applied Biosystems).
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3
PCR Protocol using GoTaq Reagents
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PCR was performed using GoTaq Green Master Mix (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The sequences of the primers used for the evaluation are listed in Table S1 . PCR products were loaded on a 2% agarose gel (Agarose S: NIPPON GENE, Tokyo, Japan) with a fluorescent DNA staining reagent (Midori Green Xtra: NIPPON Genetics, Tokyo, Japan) and separated in 1× TAE buffer. Specific bands were detected by the FAS-V gel imaging system (NIPPON Genetics).
4
Quantitative Gel Electrophoresis Analysis
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PCR and RT-PCR products were subjected to 1% or 2% agarose gel electrophoresis by using Agarose S (Nippon Gene) in 1 × TAE (40 mM Tris-HCl, 20 mM acetic acid and 1 mM EDTA, pH 8.0). When required, the relative intensity of each band was measured using ImageJ43 (link) software (National Institutes of Health, Bethesda, MD). Uncropped and unprocessed scans of the gel images presented in the figures are supplied in the Source Data file.
5
Specific Detection of E. albertii O-genotypes
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Based on the sequence variation in the wzx genes of all E. albertii Escherichia Shigella E. albertii E. albertii E. albertii
6
Detailed Inhibition Assay Protocols
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FQs, CIP, LVX, and MOX that were used in inhibition assays were purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Kanamycin and ampicillin were purchased from Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan). Restriction enzymes and lambda DNA-HindIII DNA marker were obtained from New England Biolabs, Inc. (Ipswich, MA). DNA ligation kit, Mighty Mix, and Mighty TA cloning kit were purchased from TaKaRa Bio Inc. (Shiga, Japan). Relaxed pBR322 DNA was purchased from John Innes Enterprises Ltd. (Norwich, United Kingdom). Luria-Bertani (LB) broth (Lennox) and LB agar were purchased from Sigma (St. Louis, MO, USA). Agarose S was purchased from Nippon Gene (Toyoma, Japan). Agarose I was obtained from Dojindo (Kumamoto, Japan). Gel red was obtained from Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan).
7
Comparative DNA Staining Reagents for Electrophoresis
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Midori Green Direct (Nippon Genetics), Novel Juice (Bio-Helix), Safelook Load-Green (Fujifilm Wako Pure Chemical Corporation), EZ-Vision One (VWR Life Science), and Safelook Load-White (Fujifilm Wako Pure Chemical Corporation) were used as loading dye-type of DNA-staining reagents. Midori Green Direct was supplied as 10× loading dye. Novel Juice, Safelook Load-Green, EZ-Vision One, and Safelook Load-White were supplied as 6× loading dye. SYBR Safe (Thermo Fisher Scientific, Carlsbad, CA) was used as a major nontoxic post-DNA-staining reagent. A 1Kb DNA Ladder RTU (Bio-Helix, 100 ng/μL) was used as DNA ladder marker (10, 8.0, 6.0, 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 0.75, 0.50, 0.25 kbp). Agarose S (Nippon Gene, Tokyo, Japan) was used for DNA gel electrophoresis.
8
Molecular Detection of T. b. rhodesiense
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SRA PCR was employed to identify T. b. rhodesiense using the primers described by Radwanska et al. [47 (link)] (Supplementary Table 2 ). Reagents used for each reaction included 5 μL Ampdirect plus (Shimadzu, Japan), 0.05 μL BIOTAQ HS DNA Polymerase (5 U/μL) (Bioline, UK), 0.2 μL of each 10 mM primer, 2.55 μL RNase-free water, and 2 μL extracted DNA. The temperature and cycling profile included an initial hold for 10 min at 95 °C, followed by 40 cycles at 94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 5 min. PCR products were examined by electrophoresis in 2% agarose S (Nippongene, Japan) in TAE buffer and stained using GelRed (Biotium, USA) dye before being visualized under ultraviolet light.
9
Quantifying Cell Colony Formation
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One milliliter of 0.5% agarose‐S (Nippon Gene) in DMEM was solidified in each well of a 6‐well plate or 3.5 cm dishes (Grainer Bio‐One, Nürtingen, Germany) as the bottom agarose layer and incubated for 1 h at room temperature. The top agarose (0.4%) layer containing cells was poured over the bottom agarose layer (10 000 cells per well) and set for 30 min at room temperature. One milliliter of DMEM containing 10% FCS was overlayed. The samples were cultured in a humidified incubator at 37 °C with 5% CO2 for 20 days and the overlayed medium was removed. Colonies were stained for 20 min with phosphate‐buffered saline containing 0.5% Crystal Violet. Digital images of the colonies were acquired. Colonies were manually counted.
10
Primer-Based PCR Amplification Protocol
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Primer sets used in this study are listed in S1 Table . PCR amplification was performed with Tks Gflex™ DNA Polymerase Low DNA (Takara Bio) and 0.3 μM each of the forward and reverse primers. Thermal cycling conditions consisted of one cycle at 94°C for 1 min, followed by 35 or 40 cycles at 98°C for 10 s, 60°C for 10 s, and 68°C for 24 s. Amplified PCR products were electrophoresed at 100 V for 30 min on a 1.5% (w/v) agarose gel (Agarose S; NIPPON GENE, Tokyo, Japan) containing 0.0015% (v/v) SAFELOOK Pre-Green (FUJIFILM).
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