If trace amounts of
sulfur or heavy metals will
interfere with your procedure,
the membrane should be prepared
as described below;
1. Wearing gloves,
cut the dialysis membrane into
the desired length and soak
in distilled water for 15 minutes.
2. Heat the pre-cut
membrane for 30 minutes, while
stirring, to 80 °C in a large
volume of 10 mM sodium bicarbonate.
3. Transfer the membranes
into a 10 mM Na2EDTA solution and
soak for 30 minutes.
4. Replace solution
with distilled water and stir
for 30 minutes at 80 °C.
5. Allow membrane
to cool down, and store in a
refrigerator in a 0.05% sodium
azide solution, or a 0.10% sodium
benzoate solution. Alternatively,
a 20-50% ethanol solution may
be used. Tubing must always
remain submersed.
6. Before use, wash
tubing inside and out with distilled
water and condition in dialysis
buffer (if necessary, tubing
may be sterilized).
7. Secure clamp on
one end of membrane. Buffer
or water should be placed inside
the bag to ensure integrity
of the seal. Check integrity
of the tubing and clamps.
8. Pour out test solution
and load sample (For concentrated
salt samples, leave space in
the tubing to allow for net
flow of water into the sample
and to prevent tubing from bursting.)
9. Immerse dialysis
tubing in beaker or flask containing
a large volume (usually 100
to 1000-fold that of the sample)
of the desired buffer and dialyze
for several hours at the desired
temperature with gentle stirring.
NOTE: Low-molecular weight salts
and buffers (e.g.,Tris·Cl and
KPO>4) equilibrate within
3 hours with stirring. Equilibration
times for viscous samples will
be longer.
10. Change the dialysis
buffer as necessary. Usually
two dialysis buffer changes
are sufficient. When CsCl is
removed from equilibrium density
gradient-banded DNA, two equilibrations
against a 1000-fold volume excess
of buffer will decrease CsCl
concentration 106-fold, to a
still-significant 5 μM, and
it may be necessary to change
buffer a third time.
11. Remove dialysis
tubing from buffer, remove clamp
from one end, and remove sample
with a pipet. |
