Hoefer SE640 - Manual (Page 20)

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2
Overlay each gel with a thin layer of water-saturated
n-butanol, water, or diluted gel buffer to prevent
gel exposure to oxygen. Slowly deliver the overlay
solution from a glass syringe fitted with a 22-gauge
needle. Apply the solution near the spacer at the
side of the sandwich and allow it to flow across the
surface unaided.
3
Allow the gel to polymerize for a minimum of one hour.
Stacking gel preparation
Pour the stacking gel while the sandwich is still
in the gel caster. Stacking gel resolution is opti-
mal when poured just before electrophoresis.
1
Remove the overlay by rinsing the top of the gel
several times with distilled water. Invert the caster
to drain. To ensure a seamless contact between the
resolving and stacking gels, remove residual liquid
by blotting one corner with a lint-free tissue.
2
Prepare the required amount of stacking gel monomer
solution, deaerate it, and add catalyst (APS) and
initiator (TEMED). Pour the stacking gel onto the
resolving gel with a disposable or Pasteur pipette to
a level about 2 mm from the top of the plate.
3
Introduce a comb (at a slight angle) into the sand-
wich, taking care not to trap air under the teeth. Allow
a minimum of one hour for the gel to polymerize.