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RPN2510EUM Chapter 9 Rev C 06/2007 13
Symptom
1. Membrane curling up in
hybridization bottle
2. High background
3. Weak signal
4. Patchy backgrounds
5. High background around edge of
membrane
Cause
1.1. Incorrect orientation of bottle in
rotisserie
2.1. Probe concentration too high
2.2. Unincorporated
32
P nucleotides not
removed
2.3. Insufficient blocking
2.4. Insufficient washing
3.1. No transfer from gel to membrane
3.2. Probe not labeled
3.3. Probe not denatured
3.4. Low specific activity of probe
3.5. Washes too stringent
4.1. Hybridization buffer or wash
solution not evenly covering
membrane
4.2. Damaged membrane
5.1. Damaged membrane
Remedy
1.1. Ensure membrane is rolled up in
the same direction as the bottle is
rotating (see section 7.2)
2.1. Reduce probe concentration
2.2. Remove unincorporated
nucleotides
2.3. Use recommended hybridization
buffer or extend prehybridization
time
2.4. Increase number of buffer changes
during the washing stage or
increase stringency of final wash
3.1. Load extra lanes with control DNA.
Transfer can be checked by
restaining gel with ethidium
bromide. If large DNA fragments
are detected poorly, use a
depurination step (0.25 M HCI)
3.2. Check probe labelling before
hybridization. See protocol in probe
labelling booklet
3.3. Boil probe for 5 minutes before
adding to hybridization buffer
3.4. Review labelling conditions
3.5. Increase buffer salt concentration
and decrease temperature
4.1. Increase volume of hybridization
wash or solutions or use mesh (see
section 7.1. step 4)
4.2. Handle membrane carefully with
forceps
5.1. Use clean scissors or a sharp
scalpel to cut membrane
9. Troubleshooting guide
This section briefly summarizes some of the potential problems encountered during membrane hybridizations. More
complete troubleshooting guides are supplied in the pack leaflet that accompany each product.