Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 25)

11. Stand the sandwich assembly upright on a flat surface. Loosen the comb gasket holder
screws until the threads can no longer be seen. Mark an arrow on the middle of the screw head
using a permanent marker (this will be the marker for adjusting the proper screw tension). With
the comb gasket screws and the long plate facing you, slide the comb gasket holder down over
the top of the assembled glass sandwich. When the comb gasket is properly placed, the
angled cuts on the edges of the comb gasket will rest on the complementary angled cuts at
the top of each spacer. Turn the screws until they just make contact with the glass plate, then
twist the screws an additional 1/4 turn.
12. Before the screws can be completely tightened, the pressure clamp must be attached to the
sandwich assembly. Loosen the pressure clamp screw. Mark an arrow on the middle of the
screw head using a permanent marker (this will be the marker for adjusting the proper screw
tension). Lay the pressure clamp on a flat surface so that the notched cut-out faces the
ceiling and the pressure clamp screw points up and away from you.
13. Without touching the comb gasket holder, turn the assembled glass sandwich so that the comb
gasket screws are facing down and the vent ports are facing up. Center the assembly over the
pressure clamp and allow the assembly to rest on top of it. A properly placed pressure clamp
will be situated on the middle of the sandwich with the notched cut-out against the bottom of
the glass plate. Proper placement of the sandwich assembly in the pressure clamp will insure
that equal force is applied to the comb gasket holder during the final tightening of the screws.
Twist the pressure clamp screw until it makes contact with the comb gasket holder, then
tighten the pressure clamp screw two additional turns (use the arrow to keep track of the turns).
Note: Check to insure that the bottom of the glass plates are still flush. If the plates are
off-set, one or both of the sandwich clamps may not be tightened. Repeat steps 5–13.
14. Tighten the comb gasket screws an additional one turn. If it is tightened more, the
glass plates may crack. For a proper seal, check to see that the notches on both the
comb gasket and spacers are sealed against each other. It is important that the gasket
is placed properly to prevent leakage while casting. Remove the pressure clamp.
15. Twist the injection port fitting into the holes on the sandwich clamps. Do not over-tighten; it will
damage the O-ring and cause leakage. A snug fit is all that is needed to place the injection
port against the glass plate assembly. Push a stopcock into each of the injection port fittings.
Make sure that the fit is snug. A loose stopcock may cause leakage during casting.
16. Place the gray sponge onto the front casting slot. The camshafts on the casting stand should
have the handles pointing up and pulled out. Place the sandwich on the sponge with the shorter
plate facing forward. When the sandwich is placed correctly, press down on the sandwich and
turn the handles of the camshaft down so that the cams lock the sandwich in place.
a. For a 7.5 x 10 cm dual gel sandwich, only half of the sandwich is cast at a time. Open
the stopcock and unplug the vent port on the side of the sandwich where the gel is
being cast. To prevent leakage the other half of the sandwich should have a closed
stopcock and plugged vent port.
b. For a 16 x 16 cm gel sandwich, both vent ports should be plugged to prevent leakage
during casting. The 16 x 16 cm spacers are one orientation only, thus the special
casting groove is always on the right-hand side and the smaller, shorter groove on the
left-hand side of the gel sandwich.
17. Tilt the gel sandwich assembly and casting stand using the tilt rod as a leg. Adjust the tilt
level to the highest etched line on the rod (the one farthest from the black tilt-rod cap) for
the 7.5 x 10 cm format and the lowest etched line on the rod for the 16 x 16 cm format.
18. Familiarize yourself with the Model 475 Gradient Delivery System before casting
perpendicular gradient gels.
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