Bio-Rad CHEF-DR II System - Manual (Page 14)

Section 3
Sample Preparation
3.1 Agarose Blocks
Standard procedures for DNA preparation do not yield intact, high molecular weight
DNA molecules. Large DNA molecules (chromosome-sized) are so fragile that they are
sheared by mechanical forces, such as pipetting, during isolation. To prevent breakage of
large DNA molecules, intact cells embedded in agarose are lysed and deproteinized in situ.
219
The agarose matrix protects the embedded DNA from shear forces and provides an easy way
to manipulate samples. Processed genomic DNA-embedded agarose plugs are loaded direct-
ly into sample wells of agarose gels.
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, it is not reli-
able. Different strains, plasmid content and growth media all contribute to the actual cell num-
ber 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 elim-
inate the need to generate a growth curve for each individual strain, a hemocytometer provides
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 can
be found in Section 3.7.
Sample inserts are cast in Bio-Rad’s disposable plug mold, catalog number 170-3713.
Each sample mold produces up to fifty 10 x 5 x 1.5 mm agarose plugs. The block thickness
allows rapid and efficient diffusion of enzymes during sample preparation and permits sam-
ples to be loaded into wells formed with Bio-Rad’s standard well-forming combs without
excessive trimming.
3.2 Liquid Samples
High molecular weight DNA can be prepared by standard procedures. DNA fragments of
up to several hundred kilobases do not require preparation in agarose blocks, but can be added
to the wells in liquid form. When working with DNA in the range of 50-200 kb, it may be nec-
essary to use pipette tips with large openings. When running only liquid samples, the best
resolution and sharpness of bands can be achieved by using a thin well comb (0.75 mm).
3.3 Preparation of Agarose Embedded Mammalian DNA
The buffers, enzymes, and agarose found in the following procedure are provided in the
CHEF Mammalian Genomic DNA Plug Kit (catalog number 170-3591; see Section 9 for
more information).
1. Prepare a cell suspension in isotonic saline or tissue culture medium without fetal bovine
serum. Count the cells and remove 5 x 10 7 cells for each ml of agarose plugs to be made
and place on ice. See Section 3.7 for hemocytometer usage. The 50 well plug mold makes
5 ml of agarose plugs we recommend making slightly more than 5 ml if all fifty wells
are to be utilized.
2. Prepare a 2% CleanCut ™ agarose (Bio-Rad) solution in sterile water and melt using a
microwave. Equilibrate the solution to 50 °C in a water bath.
3. Centrifuge the cell suspension at 1,000 x g for 5 minutes at 4 °C. Resuspend the cells in
one-half the final volume of plugs to made using Cell Suspension Buffer (10 mM Tris, pH
7.2, 20 mM NaCl, 50 mM EDTA) and equilibrate the cell suspension to 50 °C.
11