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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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