Bio-Rad Zeta-Probe Membranes - Manual (Page 6)

Soak one fiber pad by squeezing it while it is sub-
merged in 0.5x transfer buffer. Lay the soaked pad on
the open gel holder. Soak a piece of thick filter paper
(e.g., slab gel dryer type paper cut to the size of the
fiber pad) in the transfer buffer and place it on the fiber
pad. Place the gel on the filter paper. Hold the pre-
soaked Zeta-Probe membrane with both hands so that
the middle of the membrane is sagging or bowed
downward. Allow the middle of the membrane to con-
tact the gel first. Gradually lower the ends of the
membrane onto the gel. This process will expel most
bubbles from between the gel and the membrane. If
there are any remaining bubbles between the gel and
membrane, remove them by sliding a test tube or
extended gloved finger across the surface.
Note: Maintaining uniform physical contact between
the gel and membrane is of critical importance in
electrophoretic transfer.
Place a presoaked piece of thick filter paper on the
membrane followed by a presoaked fiber pad. Close
the gel holder and place it in the transfer cell so that the
membrane is on the anode side of the gel (red pole).
Add more 0.5x transfer buffer, if necessary, to bring the
buffer level to 1 cm below the electrode post.
6. Transfer at 80 V for 4 hours.
Note: For comprehensive electrophoretic transfer
instructions, including protocols, technical discussion,
and troubleshooting guide, refer to the Trans-Blot cell
operating manual.
7. After transfer, separate the membrane from the gel, rinse
the membrane briefly in 1x transfer buffer, and briefly blot
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1. Prepare the stock electrophoretic transfer buffer, 20x TAE or 5x
TBE.
2. Prepare gels for transfer immediately after electrophoresis:
A. Electrophoresis Under Denaturing Conditions
If gel electrophoresis was done under denaturing conditions
(e.g., agarose/formaldehyde gels), equilibrate the gel in 0.5x
transfer buffer for 10–15 min prior to electrophoretic transfer.
B. Electrophoresis Under Nondenaturing Conditions
1. Soak the gel in 0.2 N NaOH, 0.5 M NaCl for 30 min. For
polyacrylamide gels, be sure not to exceed 30 min,
since limited gel hydrolysis may occur with subsequent
swelling during transfer.
Note: Zeta-Probe membranes will bind nondenatured
nucleic acids. Therefore, denaturing is not mandatory
before transferring. Yet, after transferring, the blotted
Zeta-Probe membrane must be treated with NaOH.
Refer to the DNA alkaline fixation procedure (Section
2.7).
2. After base treatment, neutralize the gel by washing in 5x
transfer buffer two times, 10 min each. Then wash the
gel once in 0.5x transfer buffer for 10 min.
3. While gels are being equilibrated, soak the Zeta-Probe
membrane at least 10 min in 0.5x transfer buffer.
4. Fill the electrophoretic transfer cell to half full with 0.5x
transfer buffer, and circulate 4°C coolant through the
heat exchanger. If possible, place the cell on a magnet-
ic stirring plate and add a stirbar. Circulate buffer in the
cell by stirring to maintain uniform temperature during
the run.
5. Prepare the transfer assembly.
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