Tissue DNA from the liver and colon (mucosa and muscular layer) was isolated by phenol extraction as previously described.34 (link) The purified sample DNA (200–300 μg) and 8-azaadenine (40 nmol), internal standard for gas chromatography-mass spectrometry analysis were dissolved in DNAase free water and vacuum-dried in vacuum hydrolysis tube (Pierce, Rockford, IL, USA). Dried and powdered DNA were subjected to thermal and acidic hydrolysis by adding 88% formic acid and heating at 145°C for 30 minutes to preferentially release DNA bases (27). Acid hydrolysates were then re-vacuum-dried and reacted with guanase (4 milliunit/100 μg of DNA) in 20 mM phosphate buffer (pH 8.0) for an hour. Enzyme proteins were removed by centrifugation at 12,000 × g at 4°C for 12 minutes. The dried pellets were purged with pure nitrogen and sealed with short thread caps with Teflon tape (Fisher, Pittsburg, PA, USA). Mixtures of bis (trimethylsilyl) trifluoroacetamide/Acetonitrile (120 μl; 2:1, v/v) were added and subjected to derivatization at 120°C for 30 minutes.
Teflon tape
Manufactured by Thermo Fisher Scientific
Sourced in United States
Teflon tape, also known as plumber's tape or thread seal tape, is a thin, flexible, and non-adhesive strip of PTFE (polytetrafluoroethylene) material. It is designed to be wrapped around threaded pipe fittings to create a tight seal, preventing leaks and improving the connection between components.
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Lab products found in correlation
Quantifast sybr green, by Qiagen (1 mentions)
Qscript cdna supermix, by Avantor (1 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alamarblue assay, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Meridian Bioscience (1 mentions)
Primocin, by InvivoGen (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Pcr primers, by Eurofins (1 mentions)
2f peracetyl fucose, by Merck Group (1 mentions)
3fax peracetyl neu5ac, by Merck Group (1 mentions)
2 protocols using teflon tape
1
DNA Extraction and Hydrolysis Protocol
2
Cartilage Extracellular Matrix and Inflammatory Biomarkers
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3Fax-Peracetyl Neu5Ac and 2F-Peracetyl-Fucose were purchased from Merck. 96-well glass coverslip microplates were purchased from Ibidi. All lectins were purchased from Vector Labs. Human recombinant IL-1β, IL-6 and TNF-α were purchased from Peprotech. RNeasy Mini Kit and Quantifast SYBR Green were purchased from Qiagen. qScript cDNA SuperMix™ was purchased from VWR. RNase inhibitor was purchased from Bioline. PCR primers were purchased from Eurofins. Human MMP-13 ELISA kit was purchased from Abcam. Teflon™ tape, Live/Dead ® staining kit, alamarBlue ® assay, DAPI, TaqMan ® VIC ® -labelled GAPDH primer and TaqMan ® Universal PCR Master Mix, no AmpErase ® UNG were purchased from Fisher Scientific. Primocin was purchased from InvivoGen. TaqMan FAM™ labelled COL1/2 primers were purchased from Thermo Fisher Scientific. All other materials and reagents were purchased from Sigma-Aldrich unless otherwise stated.
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