pBR322 DNA plasmid (New England Biolabs, UK) in PBS (100 ng, 20 µL) was incubated with 0.5 MBq (8 µL) [201Tl]TlCl3 for upto144 h. [201Tl] TlCl3, originally formed using chloramine-T (method 6), was neutralised with Na2CO3 (0.1 M). Controls included untreated plasmid in PBS and equivalent amounts of non-radioactive [natTl]TlCl3 (Sigma). After treatment, plasmid (50 ng in PBS) was mixed with 6× loading dye (16 µL total volume), loaded onto a 0.8% agarose gel containing 10 µL GelRed Nucleic Acid stain (Biotium, USA) and run at 100 V (400 mA, 50 W) for 40 min. Gels, imaged using a GelDoc-ItTS2 310 Imager system (BioRad, UK) coupled with a Benchtop UV transilluminator (UVP) and GelCam 310, were analysed by ImageJ, measuring supercoiled (intact DNA), relaxed circular (single strand breaks) and linear band (double strand breaks) percentages within a lane (n = 3–12) [33 (link)].
Pbr322 plasmid dna
Manufactured by New England Biolabs
Sourced in United Kingdom
The PBR322 plasmid DNA is a commonly used cloning vector. It is a circular, double-stranded DNA molecule that can be used to propagate and maintain DNA sequences in bacterial cells. The PBR322 plasmid contains an origin of replication and antibiotic resistance genes, which allow for the selection and maintenance of the plasmid in host cells.
Automatically generated - may contain errors
Lab products found in correlation
Gelred nucleic acid stain, by Biotium (2 mentions)
201tl tlcl3, by New England Biolabs (2 mentions)
Nattl tlcl3, by Merck Group (2 mentions)
Geldoc itts2 310 imager system, by Bio-Rad (2 mentions)
Gelcam 310, by Bio-Rad (2 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Bca protein assay, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using pbr322 plasmid dna
1
Radiolabeling Plasmid DNA with Thallium-201
2
Radiolabeling Plasmid DNA with Thallium-201
3
Site-Specific DNA Labeling Protocol
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ODN-labeled DNA
was prepared by incubating double-stranded pBR322 plasmid DNA (100
ng/μL, New England BioLabs (NEB), Ipswich, MA), ODN-modified
AdoMet analogue AdoYnODN11 (10 μM) and M.TaqI (2.43 μM, 10 equiv of M.TaqI with respect
to 5′-TCGA-3′ recognition sequences on the plasmid)
in NEB buffer 4 (110 μL, 20 mM Tris–HCl, 50 mM KOAc,
10 mM Mg(OAc)2, 1 mM DTT, pH 7.9) at 65 °C for 1 h.
Plasmids were purified using the QIAquick PCR purification kit (QIAGEN,
Hilden, Germany) according to the instructions of the manufacturer.
Complete labeling was verified by the protection of the modified plasmid
against cleavage by the cognate restriction endonuclease R.TaqI. DNA samples were supplemented with R.TaqI (10 Units/μg DNA, New England BioLabs, Ipswich, MA), incubated
at 65 °C for 1 h, and analyzed by agarose gel (1%) electrophoresis
(0.5 × TBE buffer, 1 h, 6 V/cm, 0.01% GelRed). R.TaqI nicking instead of linearization was verified by cleavage of a
second (noncognate) restriction endonuclease (double digest). DNA
samples were supplemented with R.TaqI and/or R.NdeI (10 Units/μg DNA each, New England BioLabs, Ipswich,
MA), incubated at 37 °C for 2 h, and analyzed by agarose gel
(1%) electrophoresis (0.5 × TBE buffer, 1 h, 6 V/cm, 0.01% GelRed).
was prepared by incubating double-stranded pBR322 plasmid DNA (100
ng/μL, New England BioLabs (NEB), Ipswich, MA), ODN-modified
AdoMet analogue AdoYnODN11 (10 μM) and M.TaqI (2.43 μM, 10 equiv of M.TaqI with respect
to 5′-TCGA-3′ recognition sequences on the plasmid)
in NEB buffer 4 (110 μL, 20 mM Tris–HCl, 50 mM KOAc,
10 mM Mg(OAc)2, 1 mM DTT, pH 7.9) at 65 °C for 1 h.
Plasmids were purified using the QIAquick PCR purification kit (QIAGEN,
Hilden, Germany) according to the instructions of the manufacturer.
Complete labeling was verified by the protection of the modified plasmid
against cleavage by the cognate restriction endonuclease R.TaqI. DNA samples were supplemented with R.TaqI (10 Units/μg DNA, New England BioLabs, Ipswich, MA), incubated
at 65 °C for 1 h, and analyzed by agarose gel (1%) electrophoresis
(0.5 × TBE buffer, 1 h, 6 V/cm, 0.01% GelRed). R.TaqI nicking instead of linearization was verified by cleavage of a
second (noncognate) restriction endonuclease (double digest). DNA
samples were supplemented with R.TaqI and/or R.NdeI (10 Units/μg DNA each, New England BioLabs, Ipswich,
MA), incubated at 37 °C for 2 h, and analyzed by agarose gel
(1%) electrophoresis (0.5 × TBE buffer, 1 h, 6 V/cm, 0.01% GelRed).
4
Quantifying Endonuclease Activity in Muscle Tissue
5
Plasmid DNA Preparation and Irradiation
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pBR322 plasmid DNA (New England Biolabs) isolated from E.Coli (4361 base pairs)30 was used in this study. This cloning vector has been extensively used as a plasmid model system in irradiation studies irradiation studies38 (link),43 ,44 (link), allowing direct comparison to be made between this and earlier studies. The plasmid, in solution containing 10 mM Tris–HCl and 1 mM EDTA buffers to prevent degradation during freeze–thaw cycles, was diluted with purified water from 1000 ng/μl to 100 ng/μl. New England BioLabs quotes that ~ 90% of the plasmid is in a supercoiled (undamaged) form. Agarose gel electrophoresis confirmed that between 85 and 90% of the unirradiated plasmid was in this form.
Dry samples were prepared by pipetting 5 μl droplets of 100 ng/μl plasmid DNA directly on to the centre of Permafrost glass microscope slides (25 × 75 × 1 mm3, Thermo Fisher Scientific). The droplets were left to dry at room temperature, leaving a thin layer of DNA on the slide. Aqueous samples were held in sealed 1.5 ml Eppendorf tubes, with each tube containing 30 μl of plasmid solution at 100 ng/μl. All samples were stored at -20ºC before and after irradiation.
Dry samples were prepared by pipetting 5 μl droplets of 100 ng/μl plasmid DNA directly on to the centre of Permafrost glass microscope slides (25 × 75 × 1 mm3, Thermo Fisher Scientific). The droplets were left to dry at room temperature, leaving a thin layer of DNA on the slide. Aqueous samples were held in sealed 1.5 ml Eppendorf tubes, with each tube containing 30 μl of plasmid solution at 100 ng/μl. All samples were stored at -20ºC before and after irradiation.
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