Large-scale preparation of plasmid DNA.
Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.
Heilig
J S
JS
University of Colorado, Boulder, Colorado, USA.
Elbing
K L
KL
Brent
R
R
D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D002845:Chromatography; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D005822:Genetic Vectors / Q000302:isolation & purification*; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids / Q000302:isolation & purification*
D004247:DNA