Bio-Rad Trans-Blot® Cell - Manual (Page 19)

6. Power supply circuit is inoperative, or an inappropriate power supply was
used.
• Check the fuse. Be sure the voltage and current output of the power
supply match the needs of the blotting instrument.
7. Methanol in the transfer buffer is restricting elution.
• Reduction of methanol results in increased transfer efficiency of
proteins from the gel, but it also diminishes binding to nitrocellulose
and PVDF.
8. Gel percentage too high.
• Reduce %T (total monomer) or %C (crosslinker). A 5% C (with bis as
the crosslinker) will produce the smallest pore size gel. Decreasing from
this concentration will increase the pore size and increase transfer effi-
ciency.
Poor transfer—nucleic acid
1. Gel percentage is too high.
• Reduce the %T or %C in the acrylamide gel or reduce % agarose in an
agarose gel.
• Prior to transfer, cleave DNA in dilute 0.25 M HCl or RNA in dilute
NaOH.
2. Transfer time is too short or power conditions are too low.
• Increase the transfer time, or try high intensity transfer.
3. DNA or RNA cannot be transferred electrophoretically to nitrocellulose,
since high salt concentrations are required for efficient binding.
• Use Zeta-Probe membrane instead of nitrocellulose.
Swirls or missing bands; diffuse transfers
1. Poor contact between the membrane and the gel. Air bubbles or excess
buffer remain between the blot and gel.
• Use a test tube or pipet as a rolling pin, and roll over the membrane
carefully in both directions until air bubbles or excess buffer is removed
from between gel and membrane, and complete contact is established.
• Use thicker filter paper in the gel/membrane sandwich.
• Replace the fiber pads. Pads will compress with time, and will not hold
the membrane to the gel.
2. Power conditions are too high.
• Always check the current at the beginning of the run. The current may
be too high for a particular voltage setting. If the buffer is
prepared improperly, the conductivity may be too high, resulting in
excessive power delivered to the cell. See the power guidelines for
specific applications in Section 3.
3. The membrane is not properly wet or has dried out.
• White spots on the nitrocellulose membrane indicate dry areas where
protein will not bind. If wetting does not occur immediately by immer-
sion of the sheet in transfer buffer, heat distilled water until just under
the boiling point, and soak the membrane until completely wet.
Equilibrate in transfer buffer until ready for use.
• Because of the hydrophobic nature of PVDF, the membrane must be
prewet in methanol prior to equilibration in aqueous transfer buffer.
Follow the directions in the product insert.
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