Total RNA was extracted from cells using Transzol (TaKaRa, Japan), chloroform (Sangon, Shanghai, China), isopropyl alcohol (Sangon, Shanghai, China), DEPC water (Sangon, Shanghai, China) and 75% ethanol (Sangon, China). The yield of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and the integrity was evaluated using agarose gel electrophoresis stained with ethidium bromide.
Depc water
Manufactured by Sangon
Sourced in China
DEPC water is a type of water that has been treated with diethylpyrocarbonate (DEPC) to inactivate RNase enzymes. It is commonly used in molecular biology applications where the presence of RNase enzymes needs to be minimized.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Isopropyl alcohol, by Sangon (2 mentions)
Transzol, by Takara Bio (2 mentions)
Ethanol, by Sangon (2 mentions)
Chloroform, by Sangon (2 mentions)
Rnase free tubes, by NEST Biotechnology (2 mentions)
Rnaiso reagent, by Takara Bio (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Quantstudio 6 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Bortezomib, by AbMole (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cck 8 kit, by Vazyme (1 mentions)
Mg132, by AbMole (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Matrigel matrix, by Corning (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Engen lba cas12a cpf1, by New England Biolabs (1 mentions)
Nebuffer 2, by New England Biolabs (1 mentions)
7 protocols using depc water
1
Total RNA Extraction and Evaluation
2
Comprehensive RNA Extraction, cDNA Synthesis, and qPCR Protocol
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Total RNA was extracted with RNAiso Reagent (B9109, Takara) as described [15 (link)] and treated with DNase I to remove genomic DNA in DEPC water (B501005, Sangon Biotech). The cDNA synthesis was carried out in RNase-free tubes (401001, NEST Biotechnology) with the Transcriptor First Strand cDNA Synthesis Kit (4897030001, Roche), according to the manufacturer's instructions. Quantitative PCR (qPCR) reactions were performed using the Hieff qPCR SYBR Green Master Mix (H97410, Yeasen) in a QuantStudio 6 Real-Time PCR System (Life Technologies). Primer sequences for qPCR analysis are listed in Table 1 .
3
Comprehensive Experimental Protocols for Cell Culture Research
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M199 medium was purchased from Hyclone (SH30253.01). ECM was purchased from Sciencell (1001). Fetal bovine serum was purchased from Gibco (10099141). DMSO was purchased from sigma (D2650). Trizol Reagent was purchased from TAKARA (9108). Protein marker (P0076) and SDS-PAGE (P0690) were purchased from Beyotime. PVDF membrane was purchased from BBI (c62-393–0100). BSA kit (C102301-0002) and DEPC water (B501005-0500) were purchased from Sangon. The Matrigel matrix was purchased from CORNING (356234). CCK-8 kit was purchased from Vazyme (A311-01/02). Flow cytometry kit was purchased from Beyotime (C1062S). Collagenase I was purchased from Maokangbio (MX1001-1000MG). MG132 and bortezomib were purchased from AbMole (M1902 and M1686). 1,6-HD and 2,5-HD were purchased from Sigma-Aldrich (240117 and H11904).
4
Total RNA Extraction from Cells
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Total RNA was extracted from cells using Transzol (Takara Bio Inc., Shiga, Japan), Chloroform (Sangon Biotech, Shanghai, People’s Republic of China), Isopropyl alcohol (Sangon Biotech), DEPC water (Sangon Biotech), and 75% ethanol (Sangon Biotech). The yield of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and the integrity was evaluated using agarose gel electrophoresis stained with ethidium bromide.
5
Efficient CRISPR-Cas12a Genome Editing Protocol
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EnGen Lba Cas12a (cpf1), NEbuffer2.1, and TEbuffer were all purchased from New England Biolabs (Ipswitch, MA). All DNA sequences as well as g-RNA sequences were synthesized by Sangon Biotech (Shanghai, China) and the sequences are shown in Table S1 . The storage lifetime of cpf1 is 6 months under −20 °C, the g-RNA sequences should be stored at −80 °C no more than 1 month, and all the DNA sequences can be stored at −20 °C for 3 months. DEPC water was also obtained from Sangon Biotech. HAuCl4·4H2O (10 mg/mL) and adenosine (99.9%)were provided by Macklin biotech (Shanghai, China). Agarose, 1*TAE solution, Gelstain dye, loading buffer (6%), and marker (20–500 bp) were all purchased from Sangon Biotech (Shanghai, China). All the chemical reagents used in this work are analytical grade.
6
Quantitative Gene Expression Analysis
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Total RNA was isolated from cells by RNAiso Reagent (B9109, Takara) in DEPC water (B501005, Sangon Biotech) following by DNase I treatment in RNase-free tubes (401001, NEST Biotechnology). Reverse transcription was performed with 1 μg purified RNA using Transcriptor First Strand cDNA Synthesis Kit (4897030001, Roche) as described previously [20 (link)]. qPCR analysis was carried out using SYBR Green qPCR Master Mix (H97410, Yeasen) and a qPCR detection system (CFX384 Real-Time System, Bio-Rad) according to standard protocols. Primers are synthesized by Sangon Biotech and included in Table 1 .
7
Cell Culture and miRNA Extraction
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Two human breast cancer cell lines MB-231 and MCF10A were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MCF10A cells were cultured in DMEM/F-12 supplemented with 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin, 50 μg/mL hydrocortisone, 5% horse serum, and 1% penicillin-streptomycin. MB-231 cells were cultured in DMEM mixed with 10% FBS and 1% penicillin-streptomycin. Cells were all cultured at 37°C and under a 5% CO2 atmosphere. miRNAs extraction was performed using a miRNA extraction kit (Sagon, Shanghai, China) following protocols recommended by the manufacturer. miRNAs were dissolved in DEPC water Sangon Biotech (Shanghai, China) before fluorescent imaging detection.
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