Hoefer SE900 - Manual (Page 18)

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Prepare the Acrylamide Solution
1 See Appendix A for SDS-PAGE recipes. For casting six SE9102-1-1.0
gels, 400 ml is needed.
2 De-aerate the gel solution for 5 minutes. Add the (initiator) 10% w/v
APS and the (catalyst) 10% v/v TEMED* just prior to casting the gels.
Pour the gel solution into the channel in the back of the caster. The
solution will flow down the channel and fill the casssettes from the
bottom (Fig 15).
* The TEMED is diluted to 10% for better distribution during mixing of
the gel solutions.
3 Pour the solution as a slow, continuous stream. Try to minimize the
introduction of air bubbles into the flow.
4 Fill solution to 0.5–1 cm below the top of the glass plate to allow
room for the IPG strip and an agarose seal.
5 Overlay each gel with a thin layer of water-saturated n-butanol or
diluted gel buffer to get the best surface on the top of the gel. Slowly
apply the overlay near the gel surface from one side, taking care to
prevent mixing. Allow the overlay to flow across the surface unaided
(Fig 16).
6 Allow the gel to polymerize approximately one hour.
7 Remove the overlay by rinsing the top of the gel several times with
distilled water. Use diluted gel buffer as a final rinse.
8 Optional-storage: The second dimension gels can be temporarily stored
in the caster.
The second dimension gels can also be removed from the caster and
stored 1–2 days submerged in gel buffer before use. There is a risk
that with prolonged storage, the gels abosrb water, and the cassette
partially opens, leading to skewed dye fronts.
Fig 15. Pour the solution in a slow
continuous stream.
Fig 16. Applying overlay.
Note: A stacking gel can be used if desired,
but is not necessary.
Note: It is necessary to remove any butanol
overlays before storing the gels in the caster.
Leaving butanol in the caster can lead to
whitening and brittleness of the acrylic.
Caution! Acrylamide is a neurotoxin. Extreme
care should be used when handling and
preparing acrylamide solutions.