For MT measurements, we use a 7.9-kb DNA construct, prepared as described previously (24 (link)). The construct is generated by ligating handles (∼600 bp) with either multiple biotin or multiple digoxigenin moieties fragments to an unmodified central DNA segment 7.9 kb in length. Fluorescence intensity measurements of SYBR Gold are recorded in the presence of linear pBR322 plasmid DNA (NEB), which is produced by restriction of supercoiled circular pBR322 using restriction enzyme EcoRV (NEB) according to the protocol provided by the manufacturer. Completion of the linearization reaction was validated by agarose gel electrophoresis. Absorption, excitation and emission spectra of SYBR Gold are recorded in the presence of lambda phage DNA (NEB) due to large cuvette volumes needed, which was dialyzed against 1× phosphate buffered saline (PBS, consisting of 10 mM phosphate buffer, pH 7.4, with 137 mM NaCl and 2.7 mM KCl; Sigma-Aldrich, USA) prior to use.
Linear pbr322 plasmid dna
Manufactured by New England Biolabs
The linear pBR322 plasmid DNA is a common laboratory tool used in various molecular biology applications. It is a circular DNA molecule that has been linearized, making it suitable for techniques such as cloning, sequencing, and in vitro transcription. The core function of this product is to provide a template for DNA manipulation and analysis.
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2 protocols using linear pbr322 plasmid dna
1
DNA Construct Preparation and Fluorescence Measurements
2
Characterization of SYBR Gold Dye
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For MT measurements, we use a 7.9-kbp DNA construct, prepared as described previously (24) . The construct is generated by ligating handles (~600 bp) with either multiple biotin or multiple digoxigenin moieties fragments to an unmodified central DNA segment 7.9 kbp in length. Fluorescence intensity measurements of SYBR Gold are recorded in the presence of linear pBR322 plasmid DNA (NEB), which is produced by restriction of supercoiled circular pBR322 using restriction enzyme EcoRV (NEB) according to the protocol provided by the manufacturer. Completion of the linearization reaction was validated by agarose gel electrophoresis. Absorption, excitation and emission spectra of SYBR Gold are recorded in the presence of lambda phage DNA (NEB), which was dialyzed against phosphate buffered saline (PBS) prior to use.
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