Hoefer SE615 - Manual (Page 22)

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p19
symptom possible cause recommended action
8. No polymerization of Insufficient APS Increase both APS and TEMED by 30–50%
SDS gel (or incomplete or TEMED. APS solution is old. Make up fresh APS
polymerization.) each day.
APS stack is wet. APS is hygroscopic. Open a fresh bottle.
0
2
in gel solution. Degas at least 10 minutes.
Solutions at low Make sure all solutions are at room
temperature. temperature (20–30 °C).
TEMED is old. Use new TEMED.
9. Gel too soft. Not enough crosslinker. Crosslinker should be 2.6% C for standard
SDS gels where
%C = g bis × 100
(g monomer + g bis)
10. Gel is brittle. Too much bis. See #9 above.
11. Gel is white. Too much bis. Check concentrations of solutions.
See #9 above.
12. Gel contains swirls, Too much catalyst: Reduce both APS and TEMED by 25%.
polymerization artifacts. gel polymerized in
< 10 min.
Not enough catalyst: Increase both APS and TEMED by 50%.
gel polymerized Also see #8.
in > 50 min.
Solutions not mixed. Mix thoroughly after adding TEMED.
13. Bands are diffuse or broad. Sample doesn’t contain Use the same buffer for the sample
same buffer as as for the stacking gel.
stacking gel.
Too much TEMED Reduce concentrations by 25%.
or APS.
SDS or Use fresh solutions.
sample buffer is old.
Poor interface between Remove all liquid from the surface of the
separation gel and separation gel before adding the stacking
stacking gel. gel solutions.
14. Protein mobilities Incomplete See #8.
not consistent. polymerization.
Aged gels or acrylamide. Do not store liquid acrylamide more than
3 months. Use gels within 1–2 weeks of
casting. Use gels with stackers immediately.
Gas in gel. Degas gel solutions at least 10 minutes.
15. Heavy background Acrylamide or bis Use reagents specified as electrophoresis purity.
during silver staining. contain acrylic acid.
Water is impure. Use only double-distilled water.