Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 82)

9.2 Applications (continued)
Problem Solution
CDGE
Normal and mutant Recalculate constant denaturant from a
DNA unresolved perpendicular or parallel DGGE gel
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. Re-check acrylamide concentration.
3. Re-check 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
use or DNA spikes in gel Check shelf life date of acrylamide
solution.
2. Carefully load DNA in wells. Do not
pierce or puncture the wells.
TTGE
Normal and mutant 1. Recalculate temperature gradient
from DNA unresolved MacMelt software.
2. Use a small temperature ramp rate
(rr = 1 or 2).
3. For narrow temperature ranges (< 6°C),
use a smaller ramp rate (i.e. 1°C/hr).
4. For large temperature ranges (> 9°C),
use larger ramp rate (i.e. 3°C/hr).
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 temperature range and/or
run at lower temperatures.
78