Biology Reference
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foil as IAA is light sensitive. Pour off the IAA equilibration
solution and add 5 mL of cathode buffer to rinse off equilibra-
tion solution.
7.
Clean the glass of the fl at cassette thoroughly with ethanol and
water and lint-free wipes. Apply a small volume of deionized
water onto the glass plate along the closure. Remove the plas-
tic coversheet of a 2DGel DALT 12.5% NF precast gel (see
Note 4) and with the gel facing down, lay the gel down toward
the glass plate from the closure toward the hinge. Use a roller
to remove any air bubbles and excess liquid between the gel
and the glass plate. With the cassette laying fl at on the bench,
close the cassette and press the edge of closure tightly to ensure
the gel is placed fi rmly in the cassette.
8.
Melt the low-melt agarose in a microwave oven for 1 min on
HIGH. Pipette 5 mL of agarose into the cassette using needle
and syringe. Place the IPG strip on the cassette and push it
down to the gel with the IPG strip pusher. Remove any trapped
air bubbles in between the gel and IPG strip by pressing the
strip toward the gel, but do not damage the gel edge. Allow
agarose to set at room temperature.
9.
While agarose is gelling, set up the Ettan DALT twelve system.
Pour 7.5 L of anode buffer into the separation unit, turn the
pump on and ensure the system is in circulation. Spray 0.1%
SDS solution on the silicone rubber seals to facilitate loading
of the gel cassettes. Place a blank cassette after a gel cassette
into the sealing manifold of the unit. Ensure the seals do not
bend downwards by pulling up and down the cassette.
10. Pour a portion of the cathode buffer into the upper buffer
chamber to ensure there is a good seal between the cathodic
and the anodic reservoirs. Pour the remaining cathode buffer
in the chamber. Close the lid and run the apparatus according
to the program outlined in Table 2 .
Table 2
Second dimension electrophoresis program on the Ettan
DALT twelve
Phase
Current (mA)/gel
Power (W)/gel
Voltage (V)
Duration (h)
1
5
0.5
50
2
2
10
0.5
100
1
3
30
2.5
250
14
 
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