Biology Reference
In-Depth Information
9. Clean the glass surface of the fl at cassettes thoroughly with
ethanol and water using lint-free wipes. Apply a small volume
of ultrapure water onto the glass plate along the closure.
Remove but do not discard the plastic coversheet of a 2DGel
DALT 12.5% NF precast gel (see Note 8). With the gel facing
downwards, gently lay the gel on the glass plate fi rst at the
closure region then moving towards the hinge. Use a roller to
remove any air bubbles and excess liquid between the gel and
the glass plate. With the cassette lying horizontally on the
bench, close the fl ap and press the edge of closure tightly to
ensure the gel is fi rmly in place.
10. If molecular weight markers are required, pipette 20 mL of the
ECL Plex marker onto a small piece of fi lter paper (approxi-
mately 0.5 × 1 cm). Allow to dry at room temperature.
11. Melt the solidifi ed agarose in microwave oven. Stand the
loaded cassettes vertically using a rack. Pipette 5 mL of molten
agarose on top of the gel (between the glass plate and the plas-
tic backing) using a 10-mL syringe fi tted with a needle. Lay the
IPG strip into the molten agarose orientated such that the
lower pH end of the strip will be situated on the left-hand side
of the gel. Gently push the strip against the gel surface using an
IPG strip pusher. Remove any trapped air bubbles in between
the gel and IPG strip by pressing the strip towards the gel, but
do not damage the gel edge. Place the piece of fi lter paper
loaded with the molecular weight marker on the left-hand side
of the gel next to the acidic end of the strip. Allow the agarose
to set at room temperature.
12. Set up the Ettan DALT12 system. Pour 7.5 L of anode buffer
into the separation unit, turn the pump on, and ensure the sys-
tem is in circulation. Spray the silicone rubber seals and cassettes
with 0.1% SDS in water to facilitate loading. If running fewer
than 12 gels, insert the gel cassettes fi rst, then place the blank
cassettes at the front and rear of the unit, and, if necessary, in
between each gel. Ensure the seals are not bowed by gently
pulling up on the cassettes then pushing them down again.
13. Pour a portion of cathode buffer into the upper buffer cham-
ber. Check for leaks between the upper and lower chambers.
If the system is not leaking, pour the remaining cathode buffer
in the upper chamber. Close the lid and run the apparatus
according to the program outlined in Table 2 .
14. Cease electrophoretic separation when the dye front reaches
the bottom of the gel (approximately 16 h). Open the lid and
begin disassembling the tank by fi rstly removing the cassette/
blank at the rear or the apparatus. Rinse the cassettes thor-
oughly with deionized water. To remove the gels, lay the glass
side of the cassette on the bench and open the fl ap carefully.
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