Amersham Biosciences Hoefer DALT, User Manual, Page: 26 / 70

Casting Gradient Gels Hoefer DALT System
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Amersham Biosciences
Applying Overlay to DALT Slab Gels
The flatness of the top gel surface is a major determinant of the quality and resolution
of SDS slab gels. Imperfect gel tops can lead to irreproducible protein spot 2-D gel
patterns. A convex or tilted slab top surface can give rise to double spots, as protein at
the front and back gel surfaces starts moving at different points. To avoid this, quickly
and carefully pipette identical volumes of water-saturated n -butanol overlay solution
onto each gel. This results in very flat slab gel tops.
1. Immediately after removing the feed tube from the caster, slowly deliver 0.75 ml of
water-saturated n -butanol to the surface of each gel.
The overlay should spread evenly across the cassette with a minimum of mixing,
resulting in a smooth, flat gel top surface. Apply equal volumes of overlay to each
gel to produce gels of consistent heights.
2. Cover the top of the gel caster with plastic wrap and let the gels polymerize.
Polymerization
Allow non-gradient gels to polymerize for at least 1 hour; allow gradient gels 2 hours to
polymerize. Gradient gel polymerization should proceed from the top down. You can
observe this through the front and sides of the caster. The level of the dense displacing
solution falls farther as the gels contract upon polymerization.