Bio-Rad Model 785 Vacuum Blotter - Manual (Page 14)

5.2 Southern Hybridization Troubleshooting
The performance of the vacuum transfer will reflect in Southern hybridization
results. The table below summarizes the problems, the probable causes, and the
solutions. The term “signal” refers to the band on the Southern hybridization
autoradiograph. Refer to the Zeta-Probe
® GT manual also for specific troubleshooting.
Solutions/
Condition Probable Causes Preventions
1. No signal/ • Insufficient DNA loaded. • Load between 5-10 µg
weak signal. of DNA.
• Poor or no DNA trans- • Stain gel after it is trans-
ferred. ferred. Have positive
control lane. Both pre-
cautions are for checking
if DNA has transferred.
• 32 P-labeled probe is • Make sure probe concen-
faulty. trations and specific
activity is correct.
• Prolonged vacuum • Vacuum transfer no longer
transfer. than 90 minutes.
• Nylon membrane was • Bake membrane in 80
not baked. degree oven for 30
minutes.
2. Partial signal. • Porous membrane is • Use new porous gel
clotted.with agarose support or clean the
or salt at signal. clotted spot.
3. High background. • Uncleaned probe. • Remove all radio-isotopic
nucleotides in labeling
reaction. Can use Bio-Rad
P-30 desalting column.
• Dirty nylon membrane. • Soak nylon membrane
in 2x SSC for 5 minutes
before air drying.
• Hybridization conditions. • For Zeta-Probe membrane
use hybridization buffer
with 7% SDS + 0.5 M
NaH
2
PO
4
pH 7.2 + 1 mM
EDTA.
• Check hybridization
temperature.
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