Analysis of protocol variations on DNA yield.
A modified alkaline lysis method for preparing plasmid DNA from bacterial cells has been developed for automation implementation. The objective of this study was to develop a simplified centrifuge-based protocol that can process samples in a microwell plate. These manual experiments and parametric studies show that the alkaline lysis method can be modified significantly to enable DNA preparations to be done rapidly and reliably by an automated system. The conclusions of this study were (a) centrifugation at < 1500 g is sufficient, (b) centrifugation times need not be extended to compensate for the reduced force, (c) reactions can be done at room temperature, (d) reagent volumes can be reduced over those typically used, and (e) certain reagents can be combined to simplify the handling of fluids.
Armstrong
B
B
Institute for Development of Advanced Technology, General Atomics, San Diego, CA 92186.
Garner
H R
HR
D001331:Automation; D002498:Centrifugation; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli; D010957:Plasmids / Q000302:isolation & purification*; D013696:Temperature
D004269:DNA, Bacterial