Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 83)

DNA leaks between wells 1. Acrylamide not polymerized. Add more
TEMED and ammonium persulfate to
final concentration of 0.1%.
2. Degas acrylamide solution before
casting gel.
3. Let gel polymerize for at least 60 minutes.
4. Do not overload sample well. Reduce
sample volume.
Skewed or distorted bands, 1. Impurities in acrylamide. Filter before
use. or DNA spikes in gel Check shelf life date of acrylamide.
2. Carefully load DNA into wells. Do not
pierce or puncture wells.
Heteroduplex Analysis
Normal and mutant 1. Optimize concentration of DEM.
DNA unresolved 2. Add 15% urea to gel.
3. Adjust voltage or run time so that
samples travel at least 15 cm from well.
Air bubbles in gel Clean glass plates
Fuzzy DNA bands 1. Clean wells before use. Check for
matching comb and spacer thickness.
2. Let gel polymerize for at least 60 minutes.
Bands did not migrate far 1. Increase run time.
enough into gel 2. Decrease acrylamide concentration.
3. Increase voltage.
DNA leaks between wells 1. Acrylamide not polymerized. Add more
TEMED and ammonium persulfate to
final concentration of 0.1%.
2. Degas acrylamide solution before casting
gel.
3. Let gel polymerize for at least 60 minutes.
4. Do not overload sample well. Reduce
sample volume.
Skewed or distorted bands, 1. Impurities in acrylamide. Filter before
or DNA spikes in gel use. Check shelf life date of
acrylamide.
2. Carefully load DNA in wells. Do not
pierce or puncture the wells.
SSCP
Normal and mutant 1. Optimize running temperature.
DNA unresolved 2. Add 5–10% glycerol to gel.
3. Reduce crosslinking of acrylamide and
bis.
4. Use different concentration of buffer.
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