A hot alkaline plasmid DNA miniprep method for automated DNA sequencing protocols.
A hot alkaline lysis procedure for plasmid DNA isolation is described for the preparation of high-quality substrates for automated DNA sequence analysis. This miniprep procedure employs routine laboratory chemicals while avoiding the use of enzymes and organic solvents to produce plasmid DNA free of RNA contamination. The method is inexpensive, relatively quick and can yield over 20 micrograms plasmid DNA from a 5-ml overnight culture. Automated sequencing has a success rate of > or = 96% with plasmid substrates prepared by this method. In addition, the plasmid DNAs can be used successfully in all types of molecular biology manipulations.
Musich
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Dept. of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614-0581.
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D004269:DNA, Bacterial / Q000302:isolation & purification*; D010957:Plasmids; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial