Patient and control fibroblast cells were grown in DMEM GlutaMAX medium, supplemented with 10% FBS, PEST and 10 μg/ml uridine in 5% humidified atmosphere at 37°C. Approximately 5 × 106 cells were collected and lysed for 30 min at 42°C in lysis buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 10% SDS). An equal volume of phenol-chloroform was added, samples were mixed and centrifuged (15,000 g for 5 min, 4°C). The aqueous phase was saved and 100 mM NaCl and one volume isopropanol were added. After precipitation at -20°C for one hour, the samples were centrifuged (15,000 g, 20 min, 4°C) and the pellets were washed with 70% EtOH. The DNA was resuspended in 100 μl TE buffer. DNA concentrations were measured using the Qubit fluorometric instrument (ThermoFisher Scientific).
Qubit fluorometric instrument
Manufactured by Thermo Fisher Scientific
The Qubit fluorometric instrument is a laboratory equipment used for sensitive and accurate quantification of nucleic acids and proteins. It utilizes fluorescent dyes to detect and measure the concentration of target biomolecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem glutamax medium, by Thermo Fisher Scientific (1 mentions)
Gentra puregene cell kit, by Qiagen (1 mentions)
T7 dna polymerase, by New England Biolabs (1 mentions)
Nextseq 500 instrument, by Illumina (1 mentions)
Kapa hifi hotstart readymix, by Roche (1 mentions)
3 protocols using qubit fluorometric instrument
1
DNA Extraction from Fibroblast Cells
2
Genomic DNA Extraction from Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown in 70 ml of DMEM GlutaMAX medium (Gibco), 10% fetal bovine serum (Gibco) in 250 ml Spinner flasks (Bellco Glass Inc.). A total of 5x106 cells were collected by centrifugation for 5 min at 200 × g and washed once with PBS. Pellets were resuspended in 2 ml of lysis buffer (75 mM NaCl, 50 mM EDTA, 1% SDS, 20 mM HEPES pH 8.0, 200 μg/ml Proteinase K) and incubated at 42°C for 30 min. One volume of phenol-chloroform was added to the samples, then mixed and centrifuged at 15,000 × g for 5 min (4°C). The water phase was then transferred to a new tube for precipitation with 100 mM NaCl and 1 V of isopropanol. Samples were incubated at -20°C at least for 1 h. After precipitation, the samples were centrifuged (15,000 × g, 20 min, 4°C) and washed with 70% EtOH. Pellets were dissolved in 100 μl of TE buffer. DNA concentrations for library preparations, was measured with a Qubit fluorometric instrument (ThermoFisher Scientific). Patient and control fibroblast cells were grown in DMEM GlutaMAX medium, supplemented with 10% FBS, PEST and 50 μg/ml uridine. Between 4 and 11 × 106 cells were collected and total DNA was isolated using the Gentra Puregene Cell Kit (Qiagen) according to the manufacturer´s protocol.
3
Mapping of mtDNA and nDNA 5'-ends and ribonucleotides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 5′-ends in mtDNA and nDNA from mouse liver were mapped using 5′-End-seq by treating 1 μg of total DNA with 0.3 M KCl for 2 h at 55 °C. Ribonucleotides were mapped by HydEn-seq (30 (link)) by hydrolyzing 1 μg of total DNA with 0.3 M KOH for 2 h at 55 °C. Afterward, ethanol-precipitated DNA fragments were treated for 3 min at 85 °C, phosphorylated with 10 U of phosphatase-free T4 polynucleotide kinase (New England BioLabs) for 30 min at 37 °C, heat-inactivated for 20 min at 65 °C, and purified with HighPrep PCR beads (MagBio). Phosphorylated products were treated for 3 min at 85 °C, ligated to oligo ARC140 (30 (link)) overnight at room temperature with 10 U of T4 RNA ligase, 25% polyethylene glycol (PEG) 8000, and 1 mM CoCl3(NH3)6, and purified with HighPrep PCR beads (Mag-Bio). Ligated products were incubated for 3 min at 85 °C. The ARC76 ± ARC77 adaptor was annealed to the fragments for 5 min at room temperature. The second strand was synthesized with 4 U of T7 DNA polymerase (New England BioLabs) and purified with HighPrep PCR beads (MagBio). Libraries were PCR-amplified with KAPA HiFi Hotstart ReadyMix (KAPA Biosystems), purified, quantified with a Qubit fluorometric instrument (Thermo Fisher Scientific), and 75-base paired-end sequenced on an Illumina NextSeq500 instrument to locate the 5′-ends.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!