Total genomic DNA from 71 paired gastric tumors and adjacent normal tissues was extracted using a TIANamp Genomic DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s protocol. Total RNA was isolated from GC tissues using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions, followed by cDNA synthesis using the ReverTra Ace® qPCR RT Master Mix (TOYOBO, Tokyo, Japan). The amplification of DNA or reverse-transcribed cDNA was performed by nested PCR using the 2 × Phanta® Max Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) or TransStart TopTap DNA polymerase Kit (TransGen biotech, #AP151) according to the manufacturer’s protocols. The UL111A gene-specific primer sequences used for PCR are listed in Table S2. The PCR products were visualized on a 1% or 2% agarose gel stained with GelRed (Solarbio, Beijing, china).
Gelred
Manufactured by Solarbio
Sourced in China
GelRed is a sensitive nucleic acid stain used for detecting DNA and RNA in agarose and polyacrylamide gels. It exhibits high sensitivity and can detect as little as 0.1 ng of DNA per band.
Automatically generated - may contain errors
Lab products found in correlation
Phanta max master mix, by Vazyme (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
Chemiluminescence imager, by Bio-Rad (1 mentions)
Dna gel loading dye, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Anti cd63 antibody, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Branched polyethyleneimine, by Merck Group (1 mentions)
Pmdeta, by Merck Group (1 mentions)
Calcein am pi live dead cell double staining kit, by Solarbio (1 mentions)
Copper 1 bromide cubr, by Merck Group (1 mentions)
Glycidyl methacrylate, by Tokyo Chemical Industry (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Ethanolamine, by Merck Group (1 mentions)
5 protocols using gelred
1
Genomic DNA and RNA Extraction from Gastric Tissues
2
DNA Electrophoresis Separation Protocol
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According to the molecular weight of the separated DNA, a gel with the corresponding concentration was prepared. The solvent for the gel was 0.5×Tris-borate-EDTA (TBE), and the solute was agarose powder. GelRed (Solarbio, China) was added to the gel at a ratio of 1:10,000. DNA samples (10 ~ 20 μL) that were previously mixed with DNA gel loading dye (Thermo Scientific, US) were added to the gel, and electrophoresis was performed. The strips were visualized using a chemiluminescence imager (Bio-Rad, US) and analyzed using Image Lab Software (Bio-Rad, US).
3
Exosome Detection using Aptamer-MNP
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All synthetic DNA sequences and aptamer were purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China) and are listed in Table 1 . 10% fetal bovine serum was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD63 antibody was purchased from Abcam (Cambridge, MA, UK), and carboxylated MNPs were bought from XFNANO Co., Ltd. (Nanjing, China). The GelRed (1 × 10 000) was bought from Solarbio (Beijing, China). A549 non-small cell lung cancer (NSCLC) cells and BEAS-2B normal human bronchial epithelial cells were obtained from Procell (Wuhan, China). Ultrapure water prepared from a Millipore water purification system (a resistivity of 18 MΩ cm, Milli-Q Direct 8) was used in all the runs.
4
Serum Stability of PEGylated RA16
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Five picomoles of PEGylated RA16 and non-PEGylated RA16 mixture was incubated in 50% serum without chelating agents, for 0 min, 10 min, 1 hr, 3 hr, and 12 hr, respectively. RA16 samples after incubation were then loaded onto a 12% native PAGE gel, followed by GelRed (Solarbio, Beijing, China) staining for visualization and quantification.
5
GSDME Plasmid Transfection via PEI Nanoparticles
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Branched polyethyleneimine (PEI, Mw ∼25 kDa), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99 %), ethanolamine (EA, 98 %), ED, and copper(I) bromide (CuBr, 99 %) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Glycidyl methacrylate (GMA, 98 %), anhydrous dimethyl sulfoxide (DMSO), ethyl 2-bromoisobutyrate (EBA, 98.0 %), and deuterium oxide (D2O, 99 %) were purchased from Tokyo Chemical Industry Co., Ltd. (TCI, Tokyo, Japan), cell counting kit-8 (CCK8) was purchased from Dojindo (Tokyo, Japan). GelRed™ and Calcein-AM/PI Live/Dead cell double staining kit were purchansed from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Empty plasmid (pDNA), plasmid encoding enhanced green fluorescent protein (pEGFP), plasmid encoding GSDME protein were ordered in GenePharma Co., Ltd. (Shanghai, China). Information about antibodies used in this study was shown in Supplementary Tables 2 and 3
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