Amersham Biosciences Hoefer DALT, Руководство пользователя, Страница: 18 / 70

Casting Homogeneous (Non-Gradient) Gels Hoefer DALT System
14
Amersham Biosciences
Casting Homogeneous (Non-Gradient) Gels
WARNING Acrylamide is a neurotoxin. Always use mechanical pipettes and wear
protective gloves when working with acrylamide solutions, IPG strips
or surfaces that come in contact with acrylamide solutions.
1. Be sure the entire gel casting system is clean, dry, and free from any polymerized
acrylamide.
2. Prepare a sufficient volume of gel overlay solution (water-saturated n -butanol). You
need 0.75 ml of overlay for each cassette, or about 20 ml for a set of 25 cassettes.
3. Make up 200 ml of displacing solution (page 42).
4. Make up the gel acrylamide stock solution, without adding the 10% ammonium
persulfate (APS) or 10% N,N,N',N',-tetramethylethylenediamine (TEMED). See
“Homogeneous Gel Solutions” on page 46.
5. Load the gel caster with cassettes, separator sheets and filler blocks, if necessary.
Place a gel label in each cassette. See page 12 for directions.
6. Connect the feed tube to a funnel held in a ring-stand at a level about 12 inches
above the top of the gel caster (Figure 8). Insert the other end of the feed tube in the
grommet in the bottom of the balance chamber.
Figure 8. Setting up the
caster for a non-gradient
gel
7. Load the balance chamber with 150 ml heavy displacing solution.
8. Add the appropriate volumes of APS and TEMED only when you are ready to pour
the gel, not before.
Once these reagents are added, polymerization begins. You have about 10 minutes
before the gels begin to solidify. Vary the amount of TEMED added to control the
rate of polymerization.
9. Slowly pour the gel solution into the funnel, taking care to avoid introducing any
bubbles into the feed tube line.
Funnel
Grommet
Feed tube
Balance chamber