The DNA methylation level of the PTTG1IP promoter was analyzed in cells or tissue samples via bisulfite amplification followed by NGS. A total of 500 ng DNA was bisulfite treated using an EZ DNA Methylation Gold-kit (Zymo Research Corp., Irvine, CA, USA). The bisulfite-converted DNA was used to amplify the candidate fragment with a Takara EX Taq Hot Start Version kit (Takara Biotechnology Co., Ltd., Dalian, China). The PCR products were loaded on a 1.5% agarose gel for analysis and recovered for library construction and NGS using a MiSeq platform (Illumina, Inc., San Diego, CA, USA). The DNA methylation level of candidate fragments was determined by analyzing the NGS data. The primers for amplification were as follows: PTTG1IP forward, 5′-GTATTGTTGAAGGGTGTAGAGATG-3′ and PTTG1IP reverse, 5′-CCACCCACCAAAACTTAATAATTA-3′.
Ex taq hot start version kit
Manufactured by Takara Bio
Sourced in China, Japan, Germany
The Ex Taq Hot Start Version kit is a DNA polymerase enzyme designed for hot-start PCR amplification. It provides improved specificity and sensitivity for target DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Ex taq buffer, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Chelex 100, by Bio-Rad (1 mentions)
Takara pcr thermal cycler dice, by Takara Bio (1 mentions)
Pcr machine, by Thermo Fisher Scientific (1 mentions)
Qiasymphony sp, by Qiagen (1 mentions)
Abi 3730xl sequencing machine, by Thermo Fisher Scientific (1 mentions)
Thermo nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
4 protocols using ex taq hot start version kit
1
Analysis of PTTG1IP Promoter Methylation
2
Quantitative RT-PCR for Exon Skipping
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Total RNA was extracted from cells or frozen murine muscle sections using TRIzol (Invitrogen, Carlsbad, CA, USA). One hundred nanograms of total RNA template was used for RT-PCR with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. For one RT-PCR, 1 μL of cDNA template was mixed with 14.3 μL of water, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1.6 μL of 2.5 mM dinucleotide triphosphates (dNTPs), 2 μL 10× Ex Taq Buffer, and 0.1 μL Ex Taq horse serum from the Ex Taq Hot Start Version kit (Takara Bio, Shiga, Japan). The primer sequences used were Ex50F 5ʹ-GAGTGGGAGGCTGTAAACCA-3ʹ and Ex53R 5ʹ-ACCTGTTCGGCTTCTTCCTT-3ʹ for amplification of cDNA from exons 50–53. The cycling conditions were 95°C for 4 min, 35 cycles of 94°C for 30 s, 61°C for 30 s, 72°C for 30 s, and finally 72°C for 7 min. The intensity of PCR bands was analyzed using the ImageJ software (https://imagej.nih.gov/ij/ ; NIH, Bethesda, MD, USA), and skipping efficiency was calculated by dividing the densitometric value of the Δexon 51 plus 52 band with that of all bands (Δexon 51 plus 52 and Δexon 52).
3
Fungal Strain Identification via DNA Isolation and PCR
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DNA isolation and PCR were performed to confirm that the isolated strains were U. esculenta and identify their mating types. Isolates were cultured on YEPS agar or PDA medium and a clump of approximately 3 mm in diameter was scraped from each medium with a disposable inoculation loop and collected in a 2.0-mL tube. These tubes were frozen at –30°C until used. Six hundred microliters of TE with 50% (w/v) Chelex-100 (Bio-Rad Laboratories) and 200 μL of chloroform were added to the fungal clumps. Clumps were homogenized with a Mini Bead-Beater (WakenBtech) at 4,800 rpm for 1 min. After centrifugation at 20,400×g for 10 min, 150 μL of the supernatant was collected and an equal volume of chloroform was added and vortexed well. After centrifugation at 20,400×g for 5 min, 100 μL of the supernatant was used as the DNA extract.
Genomic PCR was performed using the Ex Taq Hot Start Version kit (TaKaRa Bio), the primers listed inTable 1 , and TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio). Thirty cycles of PCR were conducted under the following conditions according to the manufacturer’s instructions: denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 50 s. PCR products were electrophoresed on 1% agarose gel with TAE buffer, stained with ethidium bromide, and visualized with a Gel-Doc RX+ imaging system (Bio-Rad Laboratories).
Genomic PCR was performed using the Ex Taq Hot Start Version kit (TaKaRa Bio), the primers listed in
4
Lung Cancer Specimen and Cell Line Analysis
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Patient specimens and cell lines We randomly selected a cohort of ten lung cancer specimens from the Guangdong Lung Cancer Institute of Guangdong General Hospital in 2016. All samples, which were stored at −80°C after being frozen in liquid nitrogen, were assessed by two pathologists to ensure that more than 50% of the sample consisted of tumor tissue. We used nine nonsmall cell lung cancer cell lines (H460, PC9, H1650, H1975, A549, GLC82, L78, HCC827, and H2228), which were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai.
Reagents and instruments QIAsymphony DNA Mini Kit (Qiagen, Valencia, Germany); LungCarta ™ kit, PCR Accessory Set, iPLEX Pro Reagent Kit and SpectroCHIP® (Agena Bioscience, San Diego, CA, USA); H2O (Sigma-Aldrich, St. Louis, MO, USA); QIAsymphony SP (Qiagen, Valencia, Germany); Ex Taq ™ Hot Start Version Kit (Takara Biotechnology, Dalian, China); Thermo NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA); MassARRAY® Nanodispenser and MassARRAY® Analyzer (Agena Bioscience, San Diego, CA, USA); ABI 3730xl Sequencing Machine; and PCR Machine (Life Technologies, Carlsbad, CA, USA) were used.
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