High efficiency method to obtain supercoiled DNA with a commercial plasmid purification kit.

Abstract

Supercoiled state corresponds to the active form for plasmid applications. The relaxed circular form of plasmids is often inactive or poorly active. To obtain significant amounts of almost fully supercoiled DNA, we modified the standard protocol of a commercially available Qiagen plasmid purification kit. Our changes led to isolation of almost 100% of the plasmids in the supercoiled state. The modified protocol was used to purify different plasmids with consistent results. The purified plasmids maintain supercoiled state for about two months. The modified protocol is very advantageous because it allows easy DNA production with high degree of supercoiled form at low cost.

A Force-Activated Trip Switch Triggers Rapid Dissociation of a Colicin from Its Immunity Protein

“…DNA that was predominantly in the supercoiled conformation was isolated using a Hi-speed midi prep kit (Qiagen) at 4°C as described [57]. Nuclease activity was measured by addition of 30 nM E9 (final concentration) to purified DNA at a concentration of 50 µg/ml in 25 mM Tris.HCl buffer containing 1 mM MgCl 2 (pH 7.5) in the presence or absence of 50 nM Im9.…”

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways

“…Since, the latter two forms can lead to DNA repair activation ( Figure 3B ), it is crucial to minimise these structures during purification. Using a modified miniprep purification method, with all steps being carried out at 4°C, resulted in the formation of pure and homogenous supercoiled plasmid [7]. Following AID induced deamination, topological alterations are necessary for the dU to be paired with dG forming a dU:dG mismatch, which is achieved by the topoisomerases within the FE ( Figure 3C ).…”

Molecular effects on spermatozoa of Mytilus galloprovincialisexposed to hyposaline conditions

“…The plasmid DNA used for EMSA was pGEM3 DNA (2,867 bp) in the circular form. pGEM3 DNA was purified using the method described in Carbone, Fioretti, Fucci, Ausió, and Piscopo () from Escherichia coli HB 101 cells transformed with this plasmid, and analyzed by gel electrophoresis on 1% agarose gels in 89 mM Tris‐HCl pH 8.0, 2 mM EDTA, and 89 mM boric acid (TBE).…”

“…The plasmid DNA used for EMSA was pGEM3 DNA (2,867 bp) in the circular form. pGEM3 DNA was purified using the method described in Carbone, Fioretti, Fucci, Ausió, and Piscopo (2012) from Escherichia coli HB 101 cells transformed with this plasmid, and analyzed by gel electrophoresis on 1% agarose gels in 89 mM Tris-HCl pH 8.0, 2 mM EDTA, and 89 mM boric acid (TBE). 4.9 | Analysis of the effect of M. galloprovincialis PL proteins on DNA electrophoretic mobility DNA binding affinity of PL proteins from mussels exposed to the four different salinity conditions was evaluated by EMSA as described in Piscopo, Trifuoggi, Scarano, et al (2018).…”