Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 12)

Program Termination
The program in progress may be manually terminated by holding down PAUSE/START RUN
for 3– 4 seconds. A program can be terminated only while it is in the run mode; it can not be ter-
minated in PAUSE. When the program is terminated two beeps will sound, and the right display will
show OFF. Pressing PAUSE/START RUN again will start the program from the beginning.
When the program terminates under the timer control, the PAUSE/START RUN light
will go off, it will sound two beeps per second for 5 seconds, and the right display will show
OFF. The run timers will be reset and all parameters will be retained. The run parameters
may be used again as is, or further modified, and the program may be started again by press-
ing PAUSE/START RUN.
Clearing the Program
All parameters in Blocks 1, 2, and 3, can be cleared simultaneously to the default set-
tings when the program is stopped or off. Press RAISE and LOWER on the right side of the
panel for 5 seconds (it will sound 2 beeps per second).
Power Disruption
The CHEF-DR III system has a battery backed-up memory RAM that retains the current
program if the power is interrupted. If the program was in progress (not in PAUSE) when the
power went down, the program will automatically resume after 2 minutes in PAUSE mode
after power is restored. The PAUSE/START RUN light will flash during this 2 minutes.
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 during isolation. To prevent breakage of large DNA molecules,
intact cells embedded in agarose are lysed and deproteinized in situ. The agarose matrix pro-
tects the embedded DNA from shear forces and provides an easy way to manipulate sam-
ples. Processed agarose plug-DNA inserts are loaded directly into sample wells of agarose
electrophoresis gels.
The most important and difficult task in preparing cells for imbedding in agarose is to
obtain the proper cell concentration. Although optical density is frequently used, it is not reli-
able. Different cell lines or strains, plasmid content, and growth media all contribute to the
actual cell number achieved for a particular optical density. Variation in cell number will
cause the amount of DNA per agarose plug to vary greatly leading to over and/or under load-
ing of the sample. To eliminate the need to generate a growth curve for each strain, a hemo-
cytometer provides the most reproducible method for achieving the proper cell concentration
for different types of mammalian, bacterial, yeast, or fungal cells. 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.
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