Hoefer TE42 - Manual (Page 14)

•
p6
Assemble the transfer cassette
1
Pre-wet nitrocellulose or nylon membranes with
distilled water. Pre-wet PVDF or other hydrophobic
membranes in methanol. Then soak all membrane
types in transfer buffer for 2–5 minutes.
2
Open the cassette by releasing both latch tabs along
the edge opposite the hinges. Place the opened
cassette into a tray filled with at least 3 cm of trans-
fer buffer.
3
Assemble the transfer stack so that molecules
will migrate toward the membrane. For negatively
charged macromolecules (such as nucleic acids and
most proteins), build the stack on the grey half of
the cassette (and then later position the assembled
cassette in the tank so that this side faces the grey
anode (+) panel, which connects to the red lead):
Place one 3 mm-thick sponge on the opened
submerged cassette and press gently until all air is
expelled. Place one sheet of blotting paper on the
sponge, and then place the membrane on the blot-
ting paper. Place the gel—which contains a sample
that has been electrophoretically separated and
equilibrated (if required) with transfer buffer—on the
membrane. Gently roll a glass pipet or test tube over
the gel to expel trapped air between the membrane
and gel. Cover the gel with a sheet of blotting paper
and then place a sponge of the proper thickness
(see diagram on next page), again pressing gently to
expel trapped air.
Note: Always wear gloves when
handling membranes to avoid
getting fingerprints on them.
Important! Take great care in
removing all air bubbles at each
step because the presence of
air bubbles, especially between
the membrane and gel, blocks
transfer.