|Yap, Mee - UNIV OF WISC MADISON|
|Charkowsky, Amy - UNIV OF WISC MADISON|
Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 15, 2008
Publication Date: March 1, 2009
Citation: Kinscherf, T.G., Yap, M.N., Charkowsky, A.O., Willis, D.K. 2009. CHEF Procedures: A Rapid High-temperature Method for Sample Preparation, A High Voltage Hepes Buffer System and the Use of Nusieve® Agarose. Journal of Rapid Methods and Automation in Microbiology. 17(1):9-16. Interpretive Summary: Contour-clamped Homogenous Electric Field (CHEF) gel electrophoresis has gained wide acceptance as an analytical and epidemiological tool in molecular biology. A central requirement of successful CHEF analysis is the generation of largely intact chromosomal DNA as a starting material. This manuscript describes an improved method for lysing bacteria within agar plugs to produce the high-molecular weight DNA necessary for analysis. This improved method allows for the long term storage of prepared DNA and facilitates the restriction digestion on the bacterial chromosomal DNA. An improved buffers system for running the CHEF elecrophoresis that avoids degradation of DNA is also presented. This work improves and facilitates a diagnostic method important for the identification and analysis of bacterial pathogens of humans, animals, and plants.
Technical Abstract: A high temperature, low protease method of CHEF sample preparation with nuclease inactivation by diethlpyrocarbonate is described, along with a HEPES buffer system that permits high voltage CHEF electrophoresis in a CHEF-DRII apparatus.