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Qualitative and Quantitative Assessment of Plasmid DNA Isolation, Rev A

Audra Day, Colette Schneider, Lisa Ottmers, and Brandt L. Schneider Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.


Introduction
Small-scale isolation and purification of plasmid DNA from E. coli bacterial strains is a widely used and essential technique in molecular biology laboratories. With the rapid pace of molecular research, it is vitally important that plasmid DNA isolation and purification protocols and commercially available kits satisfy four criteria they should be reliable, efficient, reproducible, and cost-effective. We conducted an extensive test of Bio-Rads new Aurum miniprep and midiprep plasmid DNA isolation kits. We conclude that these kits satisfy all of the above criteria.


Methods
Bacterial strains were transformed with 1 l of 10 pg/l of various plasmids by electroporation (Bio-Rad Gene Pulser electroporator), and plated to LB plates containing 100 g/ml ampicillin (Amp). Single colonies were grown to saturation overnight in 3 ml of liquid LB containing 100 g/ml Amp for minipreps, or 30 ml of the same medium for midipreps. Densities of the cultures were determined according to the Aurum protocol, and 12 ODml* or 50 ODml of each culture were used for plasmid purification for miniprep and midipreps, respectively. Plasmid concentrations were quantitated with Bio-Rads VersaFluor fluorometer and Hoechst dye 33258 DNA quantitation kit. Plasmid quality was assessed by four criteria: restriction digests, transformation efficiency with both bacterial and yeast hosts, and sequencing. Restriction digests were done on 500 ng of plasmid in 20 l with EcoRI (NEB)
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