Bio-Rad CHEF Mapper® XA System - Manual (Page 44)

5. Mix with an equal volume of 1% low melting agarose prepared in PBS and cooled to
45 °C. Note: the agarose should be qualified for restriction digestion.
6. Transfer with a Pipetman ® or disposable pipet to plug mold former, avoiding bubbles.
7. Allow samples to harden at 4 °C or on ice for 10 minutes.
8. Transfer sample blocks to 3–5 volumes of 0.5 M EDTA, pH 9, 1% Sarcosyl, 0.5 mg/ml
Proteinase K (Boehringer-Mannheim).
9. Digest for 1–2 days at 50 °C with constant, gentle shaking. Samples may be stored in
this solution at 4 °C.
*Contributed by Dr. Bruce Birren, California Institute of Technology
Restriction Digestion of DNA In Agarose
1. Rinse samples twice in distilled water. Wash with 50x volume of TE either twice for
3 hours at room temperature or overnight at 4 °C by gently shaking or rotating in 15 or
50 ml conical bottom polypropylene tubes.
2. Optional: Residual Proteinase K may be inactivated by washing sample in TE with 1
mM PMSF (phenyl methyl sulfonyl fluoride) twice for 2 hours at room temperature prior
to final washes in TE. Step 1 is usually sufficient.
3. Digest samples with a restriction enzyme in 100 µl to 200 µl final volume, depending on
the thickness of the plug. To the sample, add 10–20 µl 10x digestion buffer as recom-
mended by the supplier of enzyme, nuclease free BSA to 0.1 mg/ml, distilled water, and
enzyme.
4. Digest agarose blocks with 2–10 U enzyme per µg DNA. Incubate 4 hours to overnight
at the appropriate temperature. Enzymes may be added twice, once at the beginning and
again partway through the incubation. This is recommended for enzymes that have short
lifetimes. (Refer to the manufacturer’s specifications.)
5. Stop the reaction by adding 1 ml 0.5 M EDTA. Incubate 5 minutes at room temperature.
6. Remove EDTA from sample. Place agarose slices into the wells of an agarose gel. It is
important that the blocks are flat against the front wall of the well; remove any trapped
bubbles. Fill the void space with 0.8% low melting agarose. Alternatively, place blocks
on each tooth of the gel forming comb, and cast gel around the comb.
7. Place gel in chamber, and equilibrate at the running temperature for 0.5 hour prior to
starting the run.
Electrophoresis and Results
The use of pulsed field electrophoresis to identify large fragments of human DNA homol-
ogous to a cDNA probe is illustrated in Figure 7.1. Human DNA was digested with two
different rare cutting enzymes and separated for 36 hours before blotting and hybridization.
With Mlu I, three bands are visible between about 450 and 600 kb. Nru I yields two bands in
this size range, one at 450 and one at 670 kb. In addition, with Nru I there is a faint hybridiza-
tion signal from the region near the top of the gel that contains unresolved large DNA. The
yeast markers indicate that the upper range of resolution of this gel is 900 kb. Thus, a hybridiza-
tion signal in this upper region could indicate partial digestion products or the presence of
fragments above 2 mb. In the stained gel, Figure. 7.2A, only the largest S. cerevisiae
chromosomes are resolved, though the S. pombe chromosomes are well separated. When the
Nru I digest is run under the same conditions, the two bands which had been well resolved in
Figure. 7.1 are visible only as a smear. However, the two larger bands are distinguishable,
one at about 2 mb and the other at 2.5 mb. Not I yields a single large band at about 2.2 mb.
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