Bio-Rad CHEF-DR II System - Manual (Page 18)

12. Maintain the plugs in 1x Wash Buffer for long term storage. However , for subsequent
restriction digestion, the EDTA concentration must be lowered. Wash the plugs to be
restricted for 30 minutes in 0.1x wash buffer or TE. See Section 3.7 for more information
on restriction digestion of plugs.
3.6 Accelerated Sample Preparation and Multiple Sample
Preparation
In many cases the above procedures can be shortened by reducing or eliminating some of
the incubation steps. For example, bacteria and yeast can be mixed with the cell wall lysing
enzyme immediately prior to casting the plug. By allowing the plug to solidify for 15 to 20
minutes at room temperature, the cell walls are sufficiently degraded to allow complete lysis
of the cells. The cell walls do not have to be completely degraded for PFGE, only enough to
allow release of the DNA is required. The plugs can be incubated in the Proteinase K buffer
after solidification, and the procedure can be shortened by 1 to 2 hours. The Proteinase K
incubations typically begin at the end of the day, and are usually allowed to proceed overnight.
This step can be reduced to 1-2 hours. Alternatively, the Proteinase K digestion can be elim-
inated by incubating the lysed cells in low salt buffers (such as TE) at elevated temperatures
(55 °C).
218 The drawback is that the DNA degrades after storage for more than a week or
two.
3.7 Restriction Enzyme Digestion of Plugs
1. Place one to three plugs per digest in a sterile 1.5 ml microcentrifuge tube, or if multiple
digests are to be done place the plugs in a 24 well microtiter plate. Incubate the plug with
1 ml of the appropriate 1x restriction enzyme buffer for about 1 hour with gentle agita-
tion at room temperature. Aspirate off the buffer and add 0.3 ml of fresh 1x enzyme
buffer. Add the restriction enzyme (50 U per 100 µl plug) and incubate 2 hours at the
appropriate temperature for the restriction enzyme.
NOTE: Digestion with multiple restriction enzymes may require very different optimal
reaction temperatures.
2. After digestion, remove the buffer and incubate in 1 ml of wash buffer or electrophore-
sis buffer for approximately thirty minutes with gentle agitation.
NOTE: If the plugs are to be stored for more than one day, remove the running buffer
from the tube and store at 4 °C. This will prevent possible diffusion of small (<100 kb)
DNA fragments out of the agarose plug.
3. Load 1/2 of a plug per well and adjust the volume if necessary on subsequent gels. In
addition, always load appropriate size standards. The concentration of cells recommend-
ed in the above procedures is optimized for a 10 mm wide well (the size of the standard-
comb provided with the CHEF-DR II system). For a smaller width well (e.g. 5 mm), the
plug should be cut in half along the long (10 mm) dimension, then trimmed appropriate-
ly to fit into the well.
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