Using Superfect transfection reagent (Qiagen), HPAEC (approximately 2×106 cells per dish, six dishes per transfection) were either mock transfected or transfected with 5 μg of pCP2-Tat101 plasmid. Twenty-four hours after transfection, cells were cross-linked with 1% formaldehyde and quenched with 1X Glycine followed by two consecutive washes with ice-cold 1X PBS. Cells were collected via scraping in 1X PBS supplemented with protease and phosphatase inhibitors and centrifuged at 3000 × g for 5 minutes. Following the manufacturer’s instructions, the Pierce Agarose ChIP Kit (Thermo Scientific) was used to precipitate and clean-up DNA bound by either control IgG (3 μg rabbit or mouse IgG provided in the kit), anti-Sp1 rabbit polyclonal (3 μg, ChIP-grade, Abcam) or Sp3 mouse monoclonal (3 μg, ChIP-grade, Santa Cruz). The purified final DNA was quantified using Qubit® 2.0 Fluorometry (Invitrogen Life Technologies).
Qubit 2.0 fluorometry
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit 2.0 fluorometer is a compact, easy-to-use instrument designed for accurate quantification of DNA, RNA, and protein samples. It utilizes fluorescent dye-based detection technology to provide highly sensitive and specific measurements.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq x ten system, by Illumina (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Superfect transfection reagent, by Qiagen (1 mentions)
Pierce agarose chip kit, by Thermo Fisher Scientific (1 mentions)
Chip grade, by Abcam (1 mentions)
Bby24m, by Next Advance (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Direct zol rna miniprep, by Zymo Research (1 mentions)
Zirconium oxide beads, by Next Advance (1 mentions)
Allprep dna rna ffpe kit, by Qiagen (1 mentions)
Blood and cell culture dna midi kit, by Qiagen (1 mentions)
Chef dr 2, by Bio-Rad (1 mentions)
Truseq stranded total rna sample preparation kit, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nucleospin blood kit, by Macherey-Nagel (1 mentions)
Nanovue plus spectrophotometry, by GE Healthcare (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Qiaamp dna ffpe tissue kit, by Promega (1 mentions)
8 protocols using qubit 2.0 fluorometry
1
Chromatin Immunoprecipitation of Sp1 and Sp3 Transcription Factors
2
Isolation and Analysis of Cranial Bone RNA
3
Profiling African Prostate Cancer
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This study utilized 45 FFPE advanced-stage, treatment-naïve primary prostate cancer and 11 NG nontumor prostate samples collected from four participating clinical sites within the CaPTC network (Fig. 1A ). In accordance with the U.S. Common Rule, the archived samples used in this study were reviewed and approved by the Institutional Review Boards of their respective clinical institutions (University of Ilorin Teaching Hospital, National Hospital Abuja, Lagos State University Teaching Hospital, Federal Medical Centre, Ahmadu Bello University, and the University of Abuja) and by the Institutional Review Board at Tuskegee University (Tuskegee, AL). Because of the retrospective nature of this study and the usage of deidentified archived samples, the review boards deemed informed written consent to be unnecessary for this study. Five 10-μm-thick curls were obtained from each block with >50% tumor and ≤50% necrosis and shipped to Q2 Solutions for DNA extraction and quality analysis. Following the manufacturer's protocol, genomic DNA and total RNA were purified using Allprep DNA/RNA FFPE kits (Qiagen). DNA quality and quantity were checked with Qubit 2.0 fluorometry (Life Technologies) and with KAPA hgDNA quantification and QC kits [Kappa Biosystems (Roche)]. DNA quality and quantity thresholds were >0.2 μg and a Q129/Q41 ratio >0.00225, respectively.
4
Whole Genome Sequencing Protocol
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To survey the genome profile, high-quality genomic DNA was extracted from the blood of the reference individual for whole-genome sequencing using the Blood and Cell Culture DNA Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For the quality control of purity, concentration, and integrity, we used Qubit 2.0 Fluorometry (Life Technologies, Waltham, MA, USA), NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and pulse-field gel electrophoresis (CHEF-DR II; Bio-Rad, California, USA), respectively. The following steps used for DNA extraction and quality control were similar. The short paired-end Illumina DNA library was constructed using the Illumina HiSeq X Ten system (RRID:SCR_016385 ) with the paired-end 350-bp sequencing strategy. After performing the sequencing and obtaining the data, the k-mer analysis of reads for the genome survey was calculated by the Jellyfish (RRID:SCR_005491 ) program with the default parameters. Additionally, the genome size, heterozygosity ratio, and repeat sequence ratio were calculated with the GenomeScope (RRID:SCR_017014 ) tool based on the k-mer frequency of 17.
5
Striatal Total RNA Extraction and Sequencing
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Total RNA was extracted from the right striatum of LID and NLID rats using an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Strand-specific libraries were prepared using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina, San Diego, CA, USA). Qubit 2.0 fluorometry (Life Technologies, Carlsbad, CA, USA) was used to quantify the purified libraries. An Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to confirm the insert size and calculate the molar concentration. The library was diluted to 10 pM and then sequenced on the Illumina HiSeq X-ten system. Library construction and sequencing were performed by Shanghai Biotechnology Corp. (Shanghai, China).
6
Canine Oncology Blood Biomarker Collection
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Before the first chemotherapeutic administration, blood samples of 29 dogs were collected for routine hematological and biochemical analyses. An aliquot of 200 μL retrieved from residual whole blood of each patient was preserved in EDTA and frozen in −80°C. DNA was extracted using the NucleoSpin Blood Kit (Macherey-Nagel) according to the manufacturer’s instructions. DNA quantification was carried out through Qubit 2.0 fluorometry (Thermo Fisher Scientific, Waltham, United States).
7
Whole Genome Sequencing of Olive Flounder
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Genomic DNA was extracted from strain WFLU12 that was isolated from a wild olive flounder [6 (link)] using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The concentration of genomic DNA was measured by Qubit 2.0 Fluorometry (Thermo Fisher Scientific, Waltham, MA, USA). The purity of DNA samples (UV A260/A280) was assessed by NanoVue Plus™ spectrophotometry (GE Healthcare Life Sciences, Chicago, IL, USA). Sequencing the whole genome of the WFLU12 strain involved Nextera XT library prep, Illumina HiSeq 2500 paired-end sequencing with 500 MB data guaranteed, PacBio 10 kb genomic library preparation, and PacBio sequencing using 1 SMRT cell.
8
FFPE Tissue DNA Extraction and Evaluation
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FFPE tissue samples were cut into 5-µm thick sections, incubated at 60°C for 30 min, de-paraffinized in three containers of xylene baths at 60°C (3×5 mins), rehydrated with decreasing alcohols (100, 95 and 70%, two washes for 5 mins each) and washed with distilled water two times for 5 mins each. Subsequently, genomic DNA was extracted from the tissue samples using the Maxwell 16 CSC DNA FFPE kit (cat. no. AS1350, Promega Corporation) or the QIAamp DNA FFPE Tissue kit (cat. no. 56404, Qiagen, Inc.), which provides an automated purification of genomic DNA from FFPE tissue samples, thereby maximizing simplicity and convenience. DNA concentration and purity were verified using Nanodrop 8000 UV–Vis spectrometry (Thermo Fisher Scientific, Inc.) and Qubit 2.0 Fluorometry (Thermo Fisher Scientific, Inc.). Degrees of DNA degradation were measured using a 200 TapeStation Instrument (Agilent Technologies GmbH) and quantitative PCR (Agilent Technologies GmbH) (38 (link)).
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