Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 81)

9.2 Applications
Problem Solution
Perpendicular DGGE
Only a single band is seen
Mix normal and mutant DNA samples
prior to loading well
Difficult to visualize hetero- 1. Increase amount of DNA (1–3 µg).
duplex and homoduplex 2. Use SYBR Green I dye agent
DNA bands (Molecular Probes, Inc.).
Unknown faint bands Impurity or non-specificity of PCR product
Poor gradient Insure that gradient delivery system is
working properly. See instructions
“S” curve appears to be shifted/cut 1. Increase upper gradient concentrations.
2. Level tilt rod after gel is cast.
Smear at top of gel Probably genomic DNA. This is OK
Parallel DGGE
Normal and mutant
1. Increase or decrease run time (time
course DNA unresolved run recommended).
2. Recalculate gradient range from
perpendicular gel or run a time course
gel.
Air bubbles in gel Clean glass plates
Fuzzy DNA bands 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. Decrease denaturant concentration.
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 into wells. Do not
pierce or puncture wells.
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