Bio-Rad CHEF Mapper® XA System - Manual (Page 42)

Section 7
Sample Preparation
7.1 Agarose Blocks and Liquid Samples
Standard procedures for DNA preparation do not yield intact, high molecular weight
DNA molecules. Large DNA molecules are so fragile that they are broken by mechanical
forces during their isolation. To prevent breakage of large DNA molecules, intact cells embedded
in agarose are lysed and deproteinized in situ. The agarose matrix protects the embedded
DNA from shear forces and provides an easy way to manipulate samples. Processed agarose-
plug DNA inserts are loaded directly into sample wells of agarose electrophoresis gels.
Sample inserts are cast in Bio-Rad’s disposable sample mold, catalog number 170-3713.
Each sample mold produces fifty 10 x 5 x 1.5 mm agarose blocks. The block thickness allows
efficient diffusion of enzymes during sample preparation and permits samples to be loaded into
wells formed with Bio-Rad’s standard well-forming combs.
To prepare samples, mix intact cells at a predetermined concentration with molten Low
Melt Preparative Grade Agarose (catalog number 162-0017). Aliquot samples into the cham-
bers of the mold and allow them to cool, then remove them from the mold and incubate with
detergents and enzymes to remove all of the cellular components from the DNA. After thor-
ough washing, cut agarose plugs to appropriate size and place in the wells of the agarose gel.
A detailed procedure for preparation of Saccharomyces cerevisiae chromosomes is given in
Section 7.2.
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 pipet 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).
7.2 Procedure for Saccharomyces cerevisiae Chromosomal DNA
This procedure is modified from Schwartz and Cantor, 1986. 153 With some modification,
this procedure will work with most yeasts.
1. Grow yeast cells at 30 °C to stationary phase in YPD media (1% yeast extract, 2% dex-
trose, 2% bactopeptone) for 24–48 hours.
2. Pellet yeast cells by centrifugation for 10 minutes at 3,000 rpm.
3. Melt 1.6% low melt agarose in sterile water. Keep warm by placing in a 50 °C water
bath.
4. Wash the cell pellet twice with cold 50 mM EDTA, pH 8.0. Centrifuge for about 5 min-
utes at 1,600 rpm.
5. Discard supernatant and resuspend the cells with the equal volume of overnight culture
used.
6. Determine the concentration of yeast by counting a diluted sample on a hemocytometer
or by plating an aliquot on a YPD plate overnight at 30 °C. A final cell concentration of
7 x 10
8 cell/ml yields enough DNA for a run. Determine the volume of cells needed along
with the number plugs to make.
7. Pipet the volume of cells into another tube, add Lyticase [Sigma] (2 mg/ml in 0.01 M
sodium phosphate containing 50% glycerol) to a final concentration of 1 mg/ml.
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