BIO RAD DCODE Manual page 77

DNA leaks between wells
Skewed or distorted bands,
or DNA spikes in gel
Heteroduplex Analysis
Normal and mutant
DNA unresolved
Air bubbles in gel
Fuzzy DNA bands
Bands did not migrate far
enough into gel
DNA leaks between wells
Skewed or distorted bands,
or DNA spikes in gel
SSCP
Normal and mutant
DNA unresolved
Buffer temperature does not
reach set temperature
Air bubbles in gel
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.
1. Impurities in acrylamide. Filter before use.
Check shelf life date of acrylamide.
2. Carefully load DNA into wells. Do not
pierce or puncture wells.
1. Optimize concentration of DEM.
2. Add 15% urea to gel.
3. Adjust voltage or run time so that
samples travel at least 15 cm from well.
Clean glass plates
1. Clean wells before use. Check for matching
comb and spacer thickness.
2. Let gel polymerize for at least 60 minutes.
1. Increase run time.
2. Decrease acrylamide concentration.
3. Increase voltage.
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.
1. Impurities in acrylamide. Filter before
use. Check shelf life date of acrylamide.
2. Carefully load DNA in wells. Do not
pierce or puncture the wells.
1. Optimize running temperature.
2. Add 5–10% glycerol to gel.
3. Reduce crosslinking of acrylamide and bis.
4. Use different concentration of buffer.
1. Use 50% ethylene glycol as chiller coolant.
2. Insure that DCode temperature controller
is set at desired buffer temperature.
3. Check that chiller is set to -20 °C.
Clean glass plates
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