Amersham Biosciences Hoefer DALT, Руководство пользователя, Страница: 39 / 70

Hoefer DALT System The DALT Blotting Kit
Amersham Biosciences
35
Figure 21. DALT
Electrophoresis Tank, with
barrier combs removed
I
MPORTANT
Fill the tank with water before turning the pump on. The pump is not
self-priming and can be damaged if run dry.
1. Fill the tank with 15 liters of water or used electrophoresis buffer.
Add the appropriate volumes of water and/or methanol to bring up to 22 liters.
2. Turn on the pump after the tank is filled.
At first, the pump blows some bubbles through the circulating flutes in the tank and
then establishes a vigorous circulating action in the tank. If the pump doesn't
“catch” and no circulation is observed, you have an airlock in the pump. See the
instructions on page 25.
3. If preparing fresh buffer, add the dry buffer mixture.
N
OTE
Even if no cooling is required for your system, use the pump to circulate the
buffer, to avoid buffer depletion at the electrodes.
Assemble the Transfer Cassette
Note Always wear gloves
when handling membranes
to avoid contamination with
skin proteins.
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. If necessary, equilibrate the gel in transfer buffer for 10–15 minutes.
3. 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 6 cm of transfer buffer.
4. Assemble the transfer stack so that molecules will migrate toward the membrane.
For negatively charged macromolecules, such as proteins coated with SDS, build the
stack on the anode (+) half of the cassette.