Hoefer SE300 miniVE - Manual (Page 32)

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problem possible cause solution
Incomplete
transfer Blank areas on
the membrane Remove all trapped air bubbles in the transfer stack; take espe-
cially great care during stack assembly to prevent air bubbles
from forming as each layer is placed.
Check electrode continuity.
Use a lower ionic strength buffer.
Molecules do
not migrate out
of gel Increase the field strength.
Increase the transfer period. (Try doubling it.)
Do not expose the gel to staining or fixing agents before transfer.
Use a thinner gel.
Reduce the gel acrylamide concentration.
For proteins, use 3.5 mM SDS (0.1%) in the transfer buffer.
Decrease the methanol in the protein transfer buffer or reduce the
amount to a minimum. Typically 10% methanol is required for
good binding to nitrocellulose membranes.
Increase the length of time DNA blots are depurinated.
Check the buffer pH. Most buffers should not be titrated; make
fresh buffer.
For native gels, increase the net charge on the protein by chang-
ing to a transfer buffer with a different pH. Lower pH (<6–7)
increases the positive charge on proteins; higher pH (>6–7)
increases the negative charge on proteins.
Diffuse band
patterns Conduct the electrotransfer immediately after electrophoretic
separation.
Shorten or eliminate the equilibration step before electrotransfer,
or conduct equilibration in the cold room.
If the transfer buffer contains methanol (≥10%), equilibrate the
gel for 30 minutes to allow it to shrink fully.
Note: Gel shrinkage may slow the migration of large molecules
out of the gel.
Take care that the gel does not shift once it contacts the
membrane.
Check that any preferred binding surface of the membrane faces
the gel.
Blotter troubleshooting