The expression vectors Bβ-wt and Bβ-mt were introduced into CHO cell lines using lipofection, as described previously [19 (link),20 (link)]. Transfected CHO cell lines were cultured in 5% CO2 at 37 °C. CHO cell lines were harvested 48 h after transfection. Total cellular RNA was extracted from cells using the QIAamp RNA Blood Mini Kit (Qiagen), and contaminated DNA was digested using Recombinant DNase I (Takara Bio Inc., Kusatsu, Japan), according to the manufacturer’s instructions. Reverse-transcriptase (RT) reactions were performed in 20 µL of a reaction mixture containing 1 µg of extracted total RNA, 2 µL of RT buffer, 5 µL of 2.5 mmol/L dNTP mixture, 1 µL of 500 µg/mL oligo dT, 0.2 µL of 0.1 mol/L dithiothreitol, and 0.5 µL of 200 U/µL Moloney murine leukemia virus (M-MLV) RT at 42 °C for 1 h. After the RT reaction, cDNA was amplified by PCR using the two pairs of primers that were used in the PCR of the fragment DNA Bβ-chain gene under the following conditions: 30 cycles at 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 2 min, preceded by 95 °C for 10 min, followed by 72 °C for 30 min. The amplified products were separated by electrophoresis on 1% agarose gels and purified from the gels using the Gene Clean II Kit (Funakoshi, Tokyo, Japan). DNA fragments were sequenced as described above using the primers that were used for the PCR of the patient’s genomic Bβ-chain gene.
Gene clean 2 kit
Manufactured by Funakoshi
Sourced in Japan
The Gene Clean II Kit is a laboratory product designed for the purification and recovery of DNA fragments from agarose gels or solutions. It provides a simple and efficient method for isolating DNA using silica-based binding and washing procedures.
Automatically generated - may contain errors
Lab products found in correlation
3500 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Dna extraction wb kit, by Fujifilm (2 mentions)
Qiaamp rna blood mini kit, by Qiagen (1 mentions)
Recombinant dnase 1, by Takara Bio (1 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Isofecal for beads beating, by Nippon Gene (1 mentions)
Bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using gene clean 2 kit
1
Bβ-chain Gene Expression in CHO Cells
2
Microbial Community Profiling via 16S rRNA Sequencing
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Total DNA was extracted from samples using ISOFECAL for Beads Beating (Nippon Gene,
Tokyo, Japan), according to the manufacturer’s protocol. To construct the 16S rRNA gene
libraries, we followed the protocols described in Ley et al. [26 (link)]. In brief, bacterial 16S rRNA genes (ca. 1300 bp)
were amplified separately for individuals and intestinal sections using the following
universal primer pair: 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1391R
(5’-GACGGGCGGTGWGTRCA-3’). The PCR conditions were 94°C for 2 min, followed by 20 cycles
of 94°C for 1 min, 55°C for 45 s, and 72°C for 2 min, with a final 20-min extension at
72°C. The amplified PCR products were pooled for each experimental group: i.e., the large
intestines of laboratory Suncus (Lab-LI), the small intestines of
laboratory Suncus (Lab-SI), the large intestines of wild Suncus
(Wild-LI), and the small intestines of wild Suncus (Wild-SI).
The PCR products were then gel-purified using a GENECLEAN II kit (Funakoshi, Tokyo, Japan)
and cloned using the pGEM-T Easy vector system (Promega, Tokyo, Japan). From each library,
more than 196 colonies containing cloned amplicons were processed for sequencing. The
plasmid inserts were sequenced using vector-specific primers, a Big Dye Terminator cycle
sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA), and a genetic analyzer
(Model 3730, 3130, Applied Biosystems).
Tokyo, Japan), according to the manufacturer’s protocol. To construct the 16S rRNA gene
libraries, we followed the protocols described in Ley et al. [26 (link)]. In brief, bacterial 16S rRNA genes (ca. 1300 bp)
were amplified separately for individuals and intestinal sections using the following
universal primer pair: 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1391R
(5’-GACGGGCGGTGWGTRCA-3’). The PCR conditions were 94°C for 2 min, followed by 20 cycles
of 94°C for 1 min, 55°C for 45 s, and 72°C for 2 min, with a final 20-min extension at
72°C. The amplified PCR products were pooled for each experimental group: i.e., the large
intestines of laboratory Suncus (Lab-LI), the small intestines of
laboratory Suncus (Lab-SI), the large intestines of wild Suncus
(Wild-LI), and the small intestines of wild Suncus (Wild-SI).
The PCR products were then gel-purified using a GENECLEAN II kit (Funakoshi, Tokyo, Japan)
and cloned using the pGEM-T Easy vector system (Promega, Tokyo, Japan). From each library,
more than 196 colonies containing cloned amplicons were processed for sequencing. The
plasmid inserts were sequenced using vector-specific primers, a Big Dye Terminator cycle
sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA), and a genetic analyzer
(Model 3730, 3130, Applied Biosystems).
3
Sequencing Genetic Variants in Fibrinogen Genes
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Genomic DNA was extracted from whole blood cells using a DNA Extraction WB kit (FUJIFILM-Wako Pure Chemical Ltd., Osaka, Japan) in accordance with the manufacturer's instructions. In order to analyze all exons and exon-intron boundaries in the Aα-, Bβ-, and γ-chain genes, 32 PCR primers were designed for DNA amplification by PCR as described previously [10, 11] (link). PCR products were purified from agarose gels using a Gene Clean II Kit (Funakoshi, Tokyo, Japan) and directly sequenced using a BigDye TM Teminator Cycle Sequencing Ready Reaction Kit and 3500 Genetic Analyzer (both from Applied Biosystems, Forster City, CA). To verify the deletion of the nucleotides or mutation detected by direct sequencing, the PCR products were subcloned into pCR ®︎ 4-TOPO plasmid vectors (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions, and the extracted subcloned plasmid vectors were sequenced as described above.
4
Genomic DNA Extraction and FGA, FGB, FGG Gene Sequencing
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Genomic DNA was extracted from whole blood cells using a DNA Extraction WB kit (FUJIFILM-Wako Pure Chemical Ltd., Osaka, Japan) in accordance with the manufacturer's instructions. To analyze all exons and exon-intron boundaries in the Aα-, Bβ-, and γ-chain genes, long-range PCR for FGA, FGB, and FGG was performed using TaKaRa LA Taq (TaKaRa Bio Inc., Otsu Japan) and the three pairs of primers, as described previously [13] . PCR products were purified from agarose gels using a Gene Clean II Kit (Funakoshi, Tokyo, Japan) and sequenced directly using a BigDye TM Terminator Cycle Sequencing Ready Reaction Kit and 3500 Genetic Analyzer (both from Applied Biosystems, Forster City, CA).
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