Amersham Biosciences Hoefer DALT, Руководство пользователя, Страница: 60 / 70

Troubleshooting Hoefer DALT System
56
Amersham Biosciences
Troubleshooting
Gel Casting
Gel caster leaks
• Apply a light film of GelSeal to the foam gasket each time the unit is used.
• Check the foam gasket for cracks or nicks and replace if necessary.
• If the stack is too tall, the front plate may not seat firmly against the gasket.
Remove filler plates or cassettes until the gasket seals.
Incomplete gel polymerization
• Use only recent stock of the highest quality reagents.
• If the dry ammonium persulfate does not crackle when water is added to it,
replace with fresh stock.
• Solutions with extreme pH values (especially acidic) may not polymerize.
• Remove oxygen from the gel environment. Degas the monomer solution
5 minutes before pouring and then overlay the gel surface with water-
saturated n-butanol.
• Adjust the gel solution temperature to a minimum of 20 °C, especially for
low% T gels.
• Increase both APS and TEMED by 30 – 50%.
• Make up fresh APS daily.
Gel is too soft, too brittle or white
• Adjust crosslinker concentration. Crosslinker should be at 2.6% C for
standard SDS gels where %C = (g bis
×
100)
÷
(g m g bis)
Gel contains swirls
• If gel polymerized in <10 min, too much catalyst. Reduce concentration of
ammonium persulfate and TEMED by 25%.
• If gel polymerized in >50 min, not enough catalyst. Increase concentration
of ammonium persulfate and TEMED by 50%.
• Make up fresh acrylamide stock solution.
Dye front curves up (smiles) at the edges.
• Fill the lower buffer chamber to the level appropriate for the run.
• Circulate coolant.
• Pre-chill the buffer.
• Decrease the current or voltage setting.
Vertical protein streaks
• IPG Strip not properly placed on gel surface. Avoid gouging separating gel
while loading strips.
• Perform iodoacetamide treatment.
• Make sure IPG strip uniformly contacts the gel surface along the entire
length of the strip.
Gels cast simultaneously are different sizes.
• Wait one minute before overlaying each gel so that the solution “settles.”
Use the same amount of overlay on each separation gel. Add the overlay
as rapidly as possible.
• Make sure the cassettes and foam sponges are not clogged with
polyacrylamide.
Gradient gels-uneven layering
• Add sucrose (15% final concentration) or glycerol (25% final concentration)
to the high-percent monomer solution.
• Add a small amount of bromophenol blue to the heavy solution to track the
gradient formation. Excessive amounts of bromophenol blue inhibit
polymerization.