Hoefer SE400 Manuale Utente Scaricare il pdf (Pagina 19)

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2.2 Acrylamide gel preparation

Table 1. Approximate monomer solution volume

required for a single gel

Gel thickness (mm)

Model 0.75 1.00 1.5

SE400 15 ml 23 ml 30 ml

SE410 23 ml 34 ml 45 ml

2.2.1 Resolving gel

Prepare the monomer solution and pour the gel.

Prepare the required amount of monomer solution,

deaerate, and add the initiator and catalyst just prior

to pouring the gel.

Pipet the solution into one corner of the sandwich,

taking care not to introduce any air bubbles. See

below for the appropriate solution level:

No stacking gel (Continuous system). Fill solution to

just below the top of the upper plate edge. If bubbles

are trapped, remove with a pipet or syringe. Introduce

a comb (at a slight angle) into each sandwich, taking

care not to trap air bubbles under the teeth.

2-gel sandwich. Pipet the solution into both sand-

wiches, filling each to the same level below the

notched edge.

Stacking gel. Fill solution to 3–4 cm below the top of

the glass plate. This height allows 1 cm of stacking

gel below the wells. Pour the gel and apply an overlay

(see step 3). After the gel is set, prepare the stacking

gel as described in the next section.

2-D electrophoresis (Discontinuous system). For the

second dimension resolving gel, fill solution to ~1.0

cm below the top of the glass plate (leave extra space

for a stacking gel, if required). One centimeter allows

enough space for the first dimension IPG strip or tube

gel and an agarose seal. (While transferring, take care

to avoid trapping air between the tube gel and slab

gel; seal the tube gel into place with agarose in elec-

trophoresis buffer.)

Note: Appendix A on page

29, lists recipes for the

Laemmli gel system.

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Hoefer SE400 Manuale Utente Pagina 19