LC-MS/MS analyses were performed as reported27 (link). Briefly, 1 μg of genomic DNA or S9.6 immunoprecipitated DNA was digested with a mixture of nuclease P1 (Roche), snake venom phosphodiesterase (Worthington) and alkaline phosphatase (Fermentas). One hundred nanograms of digested DNA were subjected to LC-MS/MS analysis. Data were normalized using internal isotopic standards for each 5mC oxidation derivative.
Alkaline phosphatase
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Alkaline phosphatase is an enzyme that catalyzes the removal of phosphate groups from various molecules, including nucleotides, proteins, and alkaloids. It is commonly used in biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Exonuclease 1, by Thermo Fisher Scientific (6 mentions)
Nuclease p1, by Merck Group (5 mentions)
T4 dna ligase, by Thermo Fisher Scientific (5 mentions)
Nuclease p1, by Roche (4 mentions)
Snake venom phosphodiesterase, by Worthington (3 mentions)
S1 nuclease, by Thermo Fisher Scientific (3 mentions)
Gmppnp, by Merck Group (3 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (3 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Venom phosphodiesterase 1, by Merck Group (2 mentions)
Dna restriction endonucleases, by Promega (2 mentions)
Plasmid purification kit, by Qiagen (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
White 384 well plate, by PerkinElmer (2 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Abcam (2 mentions)
Ascpf1 cas12a antibody, by Cell Signaling Technology (2 mentions)
Ant his6 antibody, by Abcam (2 mentions)
Alexa fluor 568 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated anti mouse igg, by Merck Group (2 mentions)
63 protocols using alkaline phosphatase
1
Quantitative Analysis of DNA Modifications
2
Quantitative Analysis of DNA Modifications
3
Small RNA Sequencing and Cloning
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Small RNAs were enriched by ExactSTARTTM Small RNA Cloning Kit (Epicentre Biotechnologies) following manufacturer’s instruction. Single-stranded small RNA was enriched from 200 μg total RNA, treated with alkaline phosphatase (Fermentas) followed by T4 polynucleotide kinase (Fermentas) to convert any possible 5′-hydroxyl and 3′-monophosphorylate ends to 5′-monophosphate and 3′-hydroxyl ends. After adding a poly(A) tail to the 3′-end and ligating a small RNA acceptor oligo containing a PCR priming site and a NotI restriction site to the 5′-monophosphate end, the modified single-strand small RNA was used as template for synthesis of first-strand cDNA with the small RNA cDNA synthesis primer, which consists of an oligo(dT) sequence, a PCR priming site and AscI (Biolabs) restriction site at its 5′-end. PCR was performed to generate the second strand of cDNA and to amplify double-stranded cDNA for subsequent cloning. Double-stranded cDNA was digested with endonucleases AscI and NotI, recovered by gel-purification, and ligated to the pre-cut pCDC1-K vector. The ligation mixture was then transformed to E. coli DH5α. Individual clones were selected and sequenced.
4
Plasmid Manipulation and Sequencing
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Restriction endonucleases (New England Biolabs), alkaline phosphatase (Fermentas) and T4 DNA ligase (Promega) were used in accordance with the manufacturer's specifications. Plasmids were maintained in the E. coli strain JM109 [12] (link) and isolated using the alkaline lysis method [13] or the QIAprep Spin MiniPrep kit (Qiagen). All primers were synthesized by Sigma-Aldrich. All sequencing was performed at the Biomolecular Resource Facility, Australian National University. PCR was carried out using the high fidelity PfuUltraII polymerase (Stratagene) for cloning and sequencing or iTaq (Scientifix) for screening.
5
Embryonic DNA Nucleoside Analysis
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DNA was purified from staged embryos by phenol chloroform extraction and subsequently digested to nucleosides using nuclease P1 (Roche), snake venom phosphodiesterase (Worthington), and alkaline phosphatase (Fermentas) and subjected to stable isotope dilution LC-MS/MS analysis as described in detail elsewhere [68 ].
6
Generating ACE2 Mutant Library via Error-prone PCR
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Error-prone PCR was performed in ACE2 residues 18-102 and 272-409 independently using GeneMorph II Random Mutagenesis Kit (Agilent). A 0.1 ng of plasmid was used as a PCR template and generated mutations with an average of about one mutation per 100 bp in 35-cycle reaction. The plenti ACE2 vector was digested with BamHI or MfeI-SacII (NEB) for ACE2 residues 18–102 or 272–409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 h and gel-purified on a Gel and PCR Clean-up kit (TAKARA). A 160 μl NEBuilder ligation reaction (NEB) was performed using 5 ng of the gel-purified inserts and 10 ng of the vector, then purified on a Gel and PCR Clean-up kit (TAKARA) and eluted by a 20 μl of distilled water. From the ligation, 400 μg (~8 μl) of the purified reaction was transformed into 100 ul of electrocompetent cells (Lucigen) and expanded according to the manufacturer’s protocol with 1500 V electroporation by ECM 399 (BTX). A 1000-fold dilution of the full transformation was plated to estimate the scale of mutant library.
7
Quantifying Transient RNA Dynamics
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Newly synthesized RNA was pulse labeled by incubating confluent 10-cm culture dishes with 2 mM bromouridine (BrU; Sigma Aldrich) for 1 hour, either under normal culture conditions, under OND, or after 1 hour of recovery from OND. To estimate background incorporations, cells were analyzed without exposure to BrU. All experimental conditions were performed in triplicate. Following BrU pulse labeling, RNA was extracted using Trizol (Ambion) following the manufacturer’s instructions and following digestion to nucleosides with nuclease P1 (Roche), U snake venom phosphodiesterase (Worthington) and alkaline phosphatase (Fermentas) as described by Kellner et al. [77 (link)]. RNA nucleosides were subjected to liquid chromatography-mass spectrometry analysis. Separation was performed on an Agilent 1290 UHPLC system equipped with a ReproSil 100 C18 column (3 μm , 4.6 × 150 mm, Jasco GmbH) maintained at 30 °C. Identification and quantification of nucleosides was performed on an Agilent 6490 triple quadruple mass spectrometer.
8
Phosphatase Treatment of Cell Lysates
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Cells were lysed in Hepes-Triton lysis buffer (Boston Bioproducts, Ashland, MA) (25 mM Hepes (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 1% Triton-X 100) containing halt protease inhibitor EDTA-free 100x and halt phosphatase inhibitor 100x (Pierce, Rockford, IL). Lysates were either not treated or treated with 20 units of alkaline phosphatase (Fermentas) at 37°C for 30 min and subjected to western blot analysis.
9
Polyadenylated RNA Processing for LC-MS/MS
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The amount of 300 ng of isolated polyadenylated RNA was further processed using RiboMinus Transcriptome Isolation Kit (Invitrogen). The eluted RNA was then digested by nuclease P1 (MilliporeSigma) followed by dephosphorylation by alkaline phosphatase (Invitrogen). The samples were then used for LC-MS/MS. For detailed procedures for sample process and mass spectrometry, refer to the previous report [10 (link)].
10
DNA Hydrolysis and Quantification
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DNA hydrolysis was performed as previously described.11 (link) Briefly, 1 μg of genomic DNA was first denatured by heating at 100 °C. Five units of Nuclease P1 (Sigma-Aldrich, St Louis, MO, USA, Cat # N8630) were added and the mixture incubated at 45 °C for 1 h. A 1/10 volume of 1 M ammonium bicarbonate and 0.002 units of venom phosphodiesterase 1 (Sigma-Aldrich, Cat # P3243) were added to the mixture and the incubation continued for 2 h at 37 °C. Next, 0.5 units of alkaline phosphatase (Invitrogen, Carlsbad, CA, USA, Cat # 18009-027) were added, and the mixture incubated for 1 h at 37 °C. Quantification was performed using a Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS) system in the multiple reaction monitoring mode as described11 (link) with some modifications. Before injection into the Zorbax Eclipse Plus C18 2.1 mm × 150 mm column (1.8 μm particle size) (Agilent, Santa Clara, CA, USA, Cat # 959759-902), the reactions were diluted 10-fold to dilute out the salts and the enzymes. Samples were analyzed on an Agilent 1290 series liquid chromatography instrument in tandem with the Agilent 6490 triple quadrupole mass spectrometer.
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