GAG accumulation was quantified with alcian blue binding assay after 6 h digestion of three constructs per sample at 60°C with 125 µg/ml papain (Sigma) in 5 mM L-cysteine-HCl (Fluka-Sigma), 5 mM Na-citrate, 150 mM NaCl, 5 mM EDTA (all AppliChem). GAG accumulation was determined by binding to alcian blue (Sigma), absorption was measured at 595 nm and quantified against chondroitin sulphate (Sigma) reference standards18 (link). Total double stranded DNA was measured for each sample after papain digestion. The amount of DNA was determined using SYBR green (Invitrogen) fluorescent assay (absorption measured at 535 nm), quantified by referring to calf thymus DNA standards (Sigma).
Calf thymus dna standard
Manufactured by Merck Group
Sourced in United States, Germany
Calf thymus DNA standard is a laboratory product that serves as a reference material for DNA quantification and analysis. It is derived from the thymus gland of calves and provides a standardized source of genomic DNA for use in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Papain, by Merck Group (2 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Chondroitin sulphate, by Merck Group (1 mentions)
Na citrate, by AppliChem (1 mentions)
Plant mini kit, by Qiagen (1 mentions)
Td 700 fluorometer, by Turner Designs (1 mentions)
Calf liver rna standard, by Merck Group (1 mentions)
Sybr green 2, by Merck Group (1 mentions)
Dimethylmethylene blue dmmb assay, by Merck Group (1 mentions)
12 well plate, by Corning (1 mentions)
Na2hpo4, by Fujifilm (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Fujifilm (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Analyzer infinite 200pro, by Tecan (1 mentions)
Dna quantitation kit, by Bio-Rad (1 mentions)
5 protocols using calf thymus dna standard
1
Quantifying GAG Accumulation and DNA Levels
2
Isolation and Quantification of Plant DNA
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Expanding leaves were collected from the Ampelouzos collection, wrapped in aluminum foil, dipped in liquid nitrogen and transferred to −80 °C till further use. Genomic DNA was isolated separately from 56 genotypes, considered representatives of each cultivar, using the Plant Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. For the initial grinding, the automated mill Tissuelyser (Rietchke-Qiagen, Maryland, USA) was employed in combination with liquid nitrogen. Once eluted, DNA was stored at 4 °C until further use. DNA was quantified employing the Hoechst 33,258 fluorescence dye (Sigma, St. Louis, MO, USA, No. B2883) on a computerized TD 700 fluorometer (Turner Designs, Sunnyvale, CA, USA) against calf thymus DNA standards (Sigma, No. D4764).
3
Muscle DNA and RNA Quantification
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The DNA content of muscle homogenates (diluted 1:8) was measured with the fluorescent dye Hoechst 33258 against a calf thymus DNA standard (Sigma-Aldrich GmbH, Steinheim, Germany) as mentioned in Rehfeldt and Walther (1997 (link)). The RNA content of muscle homogenates (diluted 1:80) was quantified fluorometrically with SYBR Green II against a calf liver RNA standard (Sigma-Aldrich GmbH, Steinheim, Germany) as published by Oksbjerg et al. (2000 ). DNA and RNA assays were performed in 96-well quartz microwell plates by using a Flx-800-I microplate fluorescence reader (Bio-Tek Instruments Inc., Bad Friedrichshall, Germany). Per animal, three and two technical replicates were performed for DNA and RNA, respectively.
4
Chondrocyte Culture and GAG Quantification
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After culture for 24 hr in 12-well plates (Corning), medium was changed and chondrocytes
were treated with or without PPS as described above for 72 hr. Cell lysates were prepared
by digesting in papain solution containing 300 µg/mlpapain (Sigma-Aldrich) in 20 mM Na2HPO4 (Wako), 1 mM EDTA and 2 mM
Dithiothreitol (Wako) at pH 6.8 for 18 hr at 60°C. The DNA content was determined by
Hoechst 33258 assay (Wako) with a calf thymus DNA standard (Sigma-Aldrich), using 350 nm
excitation and 460 nm emission filter set. The dimethylmethylene blue (DMMB) assay
(Sigma-Aldrich) was used to quantify glycosaminoglycan (GAG) contents with a chondroitin
sulphate standard (Wako) at 525 nm. Both assays were measured using a microplate reader
(Infinite M200 Pro, Tecan, Männedorf, Switzerland).
were treated with or without PPS as described above for 72 hr. Cell lysates were prepared
by digesting in papain solution containing 300 µg/mlpapain (Sigma-Aldrich) in 20 mM Na2HPO4 (Wako), 1 mM EDTA and 2 mM
Dithiothreitol (Wako) at pH 6.8 for 18 hr at 60°C. The DNA content was determined by
Hoechst 33258 assay (Wako) with a calf thymus DNA standard (Sigma-Aldrich), using 350 nm
excitation and 460 nm emission filter set. The dimethylmethylene blue (DMMB) assay
(Sigma-Aldrich) was used to quantify glycosaminoglycan (GAG) contents with a chondroitin
sulphate standard (Wako) at 525 nm. Both assays were measured using a microplate reader
(Infinite M200 Pro, Tecan, Männedorf, Switzerland).
5
Quantify DNA using Hoechst 33258 Fluorescence
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We performed DNA quantification using a DNA Quantitation Kit (Bio Rad, Hercules, CA), with Hoechst 33258 as dye. Depending on the expected concentration, we added 2–5 μl of sample to 200 μl fluorochrome solution (2 μg dye per ml). The calibration curve was generated using Calf Thymus DNA standard (Sigma–Aldrich). We measured fluorescence (excitation at 346 nm and emission at 460 nm) using a Tecan analyzer infinite 200Pro (Tecan Group Ltd, Männedorf, Switzerland).
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