Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 72)

7. Remove the alignment card. Remove the sandwich assembly from the casting stand and
check that the plates and spacers are flush at the bottom. If they are not flush, realign the
sandwich and spacers for a good seal (repeat steps 5–7).
8. When a good alignment and seal are obtained, tighten the clamp screws until it is finger-tight.
7.8 Casting PTT Gels
1. 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 assembly 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.
2. PTT samples are typically run in a discontinuous Laemmli gel. 31 Discontinuous gels
consist of a resolving or separating (lower) gel and a stacking (upper) gel. The stacking
gel acts to concentrate large sample volumes.
3. Insert a comb into the assembled gel sandwich. With a marker pen, place a mark on the
glass plate 1–2 cm below the teeth of the comb. This will be the level to which the separat-
ing gel is poured. Remove the comb.
4. Into a 50 ml tube, add the required amount of solution for casting the lower or separating gel
(Section 7.2). Add a final concentration of 0.09% (v/v) each of ammonium persulfate and
TEMED solutions. Cap the tube and mix.
5. Pour or pipette the gel solution into the sandwich until the gel solution reaches the mark
on the glass plate.
6. Immediately overlay the monomer solution with water, water-saturated isobutanol, or
t-amyl alcohol. Isobutanol or t-amyl alcohol can be applied rapidly with a Pasteur pipet
and bulb because very little mixing will occur. If water is used, it must be applied with a
needle and syringe, using a steady, even rate of delivery to prevent mixing.
7. Allow the gel to polymerize for 60 minutes. Rinse the overlay solution completely with
distilled water. This is especially important with alcohol overlays. Do not allow alcohol to
remain on the gels more than 1 hour to prevent dehydration of the top of the gel.
8. Into a 50 ml tube, add the required amount of solution for casting the upper or stacking gel
(Section 7.2). Add a final concentration of 0.09% (v/v) each of ammonium persulfate and
TEMED solutions. Cap the tube and mix.
9. Insert a comb in the gel sandwich and tilt it so the teeth are at a slight angle. This will prevent
air from being trapped under the comb teeth when pouring the gel solution (Figure 7.5).
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