DNA probes were generated by PCR using DNA primers based on the Mtb H37Rv sequences denoted in Tuberculist (34 (link)) with the addition of BamHI restriction sites for downstream cloning as needed. The PCR forward primer was labeled with [γ- 33P]-ATP using T4 DNA polynucleotide kinase (New England Biolabs). DNA fragments were then labeled by PCR (30 cycles) using Mtb genomic DNA as a template, diluted 1:3 and 1 μl DNA probe was used in each 10 μl binding reaction. Samples were electrophoresed on an 8% (29:1) non-denaturing polyacrylamide gel for 2.5 h with a constant voltage of 150 v. Gels were vacuum dried, exposed on a phosphor screen, scanned with a Storm 860 PhosporImager (Molecular Dynamics), and analyzed with ImageQuant software (Molecular Dynamics).
T4 dna polynucleotide kinase
Manufactured by New England Biolabs
Sourced in United States
T4 DNA polynucleotide kinase is an enzyme that catalyzes the transfer of a phosphate group from ATP to the 5' hydroxyl terminus of DNA or RNA molecules. This enzyme is commonly used in molecular biology applications that require the phosphorylation of nucleic acid fragments.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant software, by GE Healthcare (2 mentions)
Storm 860 phosporimager, by GE Healthcare (2 mentions)
Whatman, by Cytiva (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Fragment analyzer, by Agilent Technologies (2 mentions)
T4 dna ligase buffer, by New England Biolabs (2 mentions)
Klenow dna polymerase, by New England Biolabs (2 mentions)
Ligase buffer, by New England Biolabs (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Klenow fragment, by New England Biolabs (2 mentions)
T4 dna polymerase, by New England Biolabs (2 mentions)
33p atp, by PerkinElmer (1 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions)
Tag dna polymerase, by Thermo Fisher Scientific (1 mentions)
Whatman paper, by GE Healthcare (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
8 protocols using t4 dna polynucleotide kinase
1
Radiolabeled DNA Probes for Mtb Genomic Analysis
2
NF-κB p65 Binding Assay Protocol
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PCR primers were labeled with [-33P]-ATP (Perkin Elmer, Waltham, MA, USA) using T4 DNA polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Subsequently, DNA probes were created by labeling the forward primers and unlabeling the reverse primers via PCR. Two micrograms of nuclear protein were incubated with a biotin-labeled NF-κB p65 binding-site DNA probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′; Sigma Genosys) in buffer for 30 minutes on ice. Gels were placed on Whatman paper, vacuum-dried, and exposed to a phosphoscreen for 12 hours. Subsequently, the screen was scanned using a storm 860 PhosphorImager, and the data were analyzed using ImageQuant software (Molecular Dynamics/GE Healthcare, Amersham, UK).
3
Heat Shock Response in HEK293 Cells
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HEK293 cells were heat-shocked at 42 °C for 0 or 5 min and washed once with cold 1 × PBS before being collected by scraping. Genomic DNA was prepared using PureLink Genomic DNA Mini Kit (Life Technologies). Each primer was labelled with [α-32P] dATP using T4 DNA polynucleotide kinase (New England Biolabs). For primer extension, 1 μg of genomic DNA was mixed with about 4.5 × 106 c.p.m. per sample of labelled primer, 10 μM of each dNTP and thermostable, non-proofreading Tag DNA polymerase (1 unit, Invitrogen) in 25 μl of 10 mM Tris-HCl (pH 8.3), 50 mM KCl and 1.5 mM MgCl2. PCR cycles are 30 cycles of 1 min 20 s at 95/60/72 °C, followed by a final extension for 15 min at 72 °C. PCR products were separated on denaturing polyacrylamide gels and exposed to X-ray film. The primer sequences are available in Supplementary Table 1 .
4
DNA Library Preparation for Sequencing
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Purified DNA (see previous section) was quantified by Fragment Analyzer (Agilent). 25 ng of purified DNA was resuspended up to 50 μl in water. 10 μl of T4 DNA ligase buffer (NEB, B0202), 4 μl of 10 mM dNTPs, 5 μl of T4 DNA polymerase (NEB, M0203), 1 μl of Klenow DNA polymerase (NEB, M0210), 5 μl of T4 DNA polynucleotide kinase (NEB, M0201), and 25 μl of water was added to the diluted input DNA and incubated at 25°C for 30 min. Samples were purified with Ampure XP beads (Beckman, A36881) and resuspended in 32 μl of water. 5 μl of buffer 2 (NEB, B7002), 1 μl of 10 mM dATP, 3 μl of Klenow fragment (NEB, M0212), and 9 μl of water was added to the end-repaired DNA and incubated at 37°C for 30 min. Samples were purified with Ampure XP beads (Beckman, A36881) and resuspended in 23 μl of water. 5 μl of Truseq Y adaptors for paired-end sequencing (custom-made), 5 μl of 10× ligase buffer (NEB, B0202), 1.5 μl of T4 DNA ligase (NEB, M0202), and 15.5 μl of water was added to the 3’-adenylated DNA and incubated at room temperature for 1 h. Samples were purified with Ampure XP beads (Beckman, A36881) and resuspended in 30 μl of water. 3 μl of adaptor ligated DNA was used for PCR amplification (KAPA HiFi master mix, KK201).
5
DNA-Protein Binding Assay Protocol
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PCR forward primers (Supplementary Table S3) were labeled with [γ- 33P]-ATP (MP Biomedicals or Perkin Elmer) using T4 DNA polynucleotide kinase (New England Biolabs). DNA probes were generated using labeled forward primer and unlabeled reverse primer in PCR reaction. About 0.05 pmol DNA probe was used in each 10 μl binding reaction mixture. Briefly, 0.3 μM His-Cmr (N- or C-terminal tagged) and DNA probes were incubated at room temperature for 30 min in DNA binding buffer [10 mM Tris–HCl (pH 8.0), 50 mM KCl, 1 mM EDTA, 50 μg/ml bovine serum albumin, 1 mM dithiothreitol, 0.05% non-ionic P-40 detergent, 20 μg/ml poly(dI-dC) and 10% glycerol], with or without 100 μM cAMP. Samples were loaded on an 6, 8 or 12% non-denaturing polyacrylamide gel, depending on the size of the DNA probe, and run for 2–3 h at 14 V/cm in 0.5× Tris-borate-EDTA buffer at 4°C. Gels were transferred to Whatman paper, vacuum-dried, exposed overnight on a phosphor screen, scanned with Storm 860 PhosporImager (Molecular Dynamics) and analyzed with ImageQuant software (Molecular Dynamics). About 200–500× excess competitor DNA was used wherever mentioned.
6
Oligonucleotide Preparation for RSS Assays
7
DNA Library Preparation for Sequencing
8
Electrophoretic Mobility Shift Assay
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Complementary oligonucleotides were synthesized and HPLC-purified by Sigma-Aldrich. Oligomer duplexes were prepared by mixing 100 pmol of each of the two complementary oligonucleotides in a total volume of 50 µl of annealing buffer (10 mM Tris HCl pH 8.0, 5 mM MgCl2, 65 mM KCl 0.5 mM EDTA, 1 mM DTT). After 3 min incubation at 95 °C, the samples were left to form oligomer duplexes while gradually cooling to room temperature. dsDNA oligomers were 5′-32P-labeled with T4 DNA polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (Perkin Elmer, 3,000 Ci/mmol) followed by removal of the excess of radioactive ATP using LiCl/ethanol precipitation. Recombinant p651-306 protein (0.5 µM) was incubated in EMSA buffer containing 10 mM Tris HCl pH 7.5, 0.5 mM EDTA, 65 mM KCl, 5 mM MgCl2, 1 mM DTT, 100 μg/mL BSA, 10% glycerol, 50 ng/µl poly(dI-dC), and 32P-labeled ds oligonucleotide (2κB dsDNA: 5′-AATTCGGGACTTTCCCGTCGGGACTTTCC-3′; κBmut dsDNA: 5′-AGCTCGCTATTAGACAGCTGCTATTAGACTGCA-3′) for 45 min at 30 °C. Protein-DNA complexes were resolved by native 4% PAGE and visualized by autoradiography.
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