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5 Casting gradient gels
5.3 Pouring gel solutions for gradient gels
slide
stop
Fig 5-1. DALTsix gradient maker.
5.3
1
2
3
4
5
6
7
8
9
10 When the gels have reached the desired height and before air is introduced, stop the peristaltic pump. It is
16
reservoir (back)
chamber
slide
valve
Pouring gel solutions for gradient gels
Prepare the gel caster, as described on page 6, and place identifying gel labels inside each cassette.
When you are ready to cast the gels, add the APS and TEMED and mix each gel solution thoroughly. Vary
the amount of TEMED added to control the rate of polymerization. Once these reagents are added,
polymerization begins. You have about 10 min to cast the gradient before the gels begin to solidify at the top.
Work rapidly and carefully.
Close the slide valve (out on side of white slide stop button, Fig 5-1). If the outlet tubing is not controlled by a
pump, clamp it off near the gradient former. Add the required volume of the final solution to the reservoir
(back) chamber.
Carefully open the slide valve (valve in on button side) and allow just enough solution to flow through the
connector channel to fill it to the edge of the mixing chamber, then close the valve. Be sure no large bubbles
remain to obstruct flow through the channel.
Add the required volume of the light solution to the mixing chamber and start the magnetic stirrer.
Simultaneously, open the outlet tubing if clamped off, and start the pump. Adjust the pump rate so that the
solution is not forced up in a "fountain" that mixes with the overlying solution.
Immediately open the connecting valve between the mixing and the reservoir chamber.
Watch the gradient solution enter the caster. It is important that no bubbles disturb the gradient-watch the
delivery carefully.
Just as the last of the gradient mix is pumped out of the mixing chamber, add 200 ml of displacement
solution to the mixing chamber and pump it through until almost all of the gradient mix has entered the
cassettes. It is convenient to include a dye in the displacement solution to visually track the boundary
between the gradient mix and the displacement solution.
important that no air bubbles disturb the gradient.
connector channel
mixing (front)
chamber
outlet
connector
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
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