Bio-Rad CHEF Mapper® XA System - Manual (Page 43)

8. Add equal volume of 1.6% low melt agarose to the cell/enzyme suspension to give a final
agarose concentration of 0.8%.
9. Mix by inverting the tube several times. Keep the tube at 50 °C.
10. Using a disposable transfer pipet, pipet the sample into the plug molds. Allow the agarose
plugs to solidify at room temperature for about 30–45 minutes or plate at 4 °C for 20
minutes. Each well in Bio-Rad’s disposable plug mold holds approximately 100 µl of
sample.
11. Push the plugs out into a 50 ml Falcon tube containing ESP buffer (0.5 M EDTA, pH
9.0, 1 M Tris, pH 9.0, 10% N-Laurylsarcosine, 80 mg/ml Proteinase K. Take up desired
volume with 0.5 M EDTA, pH 9.0), using the provided snap-off tool on the plug mold.
12. Incubate the samples at 50 °C overnight (16–24 hours).
13. Aspirate off the ESP buffer and add enough volume to immerse the plugs with TE buffer,
pH 9.0. Let sit at room temperature for about 1 hour.
14. Decant the TE, pH 9.0 buffer and add fresh TE, pH 9.0 buffer. Let sit at room tempera-
ture for about 1 hour. Repeat wash two more times.
15. Store plugs in TE, pH 9.0 buffer at 4 °C for up to 1 year.
7.3 Procedure for Mammalian DNA*
An important application of pulsed field electrophoresis is the analysis of human and other
mammalian DNA. With this technique, fragments which are hundreds or thousands of kilobases
in length can be separated. Such fragments may contain entire gene families. Since mammalian
chromosomes are too large to enter gels with current technology, they must be digested with
restriction enzymes and visualized by probe hybridization. This section describes procedures
for preparing samples, restriction digestion, setting switch time parameters, and using appro-
priate controls. The DNA employed is from human white cells. Additional information on
sample preparation and pulsed field electrophoresis conditions is given in the references.
Sample Preparation
The following procedure will give a final concentration of 10 7 cells/ml in the agarose.
Typical agarose slices contain 50–100 µl, giving 5–10 µg DNA per lane.
1. Harvest cells in phosphate-buffered saline:
A. Collect tissue culture cells by scraping or trypsination, followed by centrifugation.
Centrifuge suspension cultures directly, although brief trypsin digestion (0.25% in
saline) can improve lysis.
B. Prepare white blood cells from whole blood by either isotonic lysis or centrifugation
through Ficoll (e.g., Sigma Histopaque ® ). Starting volume is 5–50 ml blood.
C. Mince tissue with scissors or a razor blade and disperse to single cells with a loose
fitting Dounce homogenizer. Let large clumps of tissue settle, transfer supernatant,
and centrifuge.
2. Wash cells twice with PBS at 4 °C.
3. Count cells, and resuspend in PBS at a concentration of 2 x 10 7 cells/ml. Cells may be
counted in a hemocytometer (e.g., Reichert Inc.).
4. Gently pipet up and down to break up clumps, and warm to 37 °C.
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