Bio-Rad CHEF Genomic DNA Plug Kits - Manual (Page 3)

Section 1
Introduction
Pulsed-Field Gel Electrophoresis (PFGE) allows the
separation of DNA ranging in size from a few kilobase pairs
to 10 megabase pairs. Because of the large size of these
molecules, simple pipetting mechanically shears the DNA
resulting in unacceptable quality for PFGE separations. This
has necessitated procedures for lysis of whole cells embedded
in agarose, allowing purification of chromosome-sized DNA
without shearing.
The most important and difficult task in preparing cells
for embedding in agarose is to obtain the proper cell
concentration. Although optical density is frequently used to
determine cell concentration, it is not reliable. Different
strains, plasmid content, and growth media all contribute to
the actual cell number achieved for a particular optical
density. Variation in cell number will cause the amount of
DNA per agarose plug to vary, leading to over- and or under-
loading of the sample. To eliminate the need to generate a
growth curve for each strain, a hemocytometer is the most
reproducible method for achieving the proper cell
concentration for different types of cells, bacteria, yeast, and
fungi. Detailed instructions for the use of a hemocytometer
are given in the Appendix.
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