Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus User Manual
Page 16
The carrier DNA used with nitrocellulose must be denatured before adding it to the
prehybridization solution. Heat the DNA at 100°C for 5 minutes and cool rapidly.
3.
Seal the top of the bag and incubate.
For DNA or RNA Bound to
For DNA Bound to
For RNA Bound to
Zeta-Probe Membrane
Nitrocellulose
Nitrocellulose
5 minutes at 65°C
2–4 hours at 68°C
8–20 hours at 42°C
Hybridization
1.
Cut one corner of the plastic bag. Remove the prehybridization solution and replace it with a
fresh batch of the same solution, except when binding RNA to nitrocellulose. In that case,
add 10% dextran sulfate to the hybridization solution.
Note: Formamide can also be used in the hybridization buffer to lower the incubation
temperature when binding DNA to nitrocellulose or Zeta-Probe membrane (Maniatis et al.
1982, Casey and Davidson 1977). For alternative protocols, see the Zeta-Probe membrane
instruction manual.
2.
Add the denatured probe, remove all air bubbles and reseal the bag. Mix the contents of the
bag. Hybridize with agitation.
For DNA or RNA Bound to
For DNA Bound to
For RNA Bound to
Zeta-Probe Membrane
Nitrocellulose
Nitrocellulose
4–24 hours at 65°C
4–24 hours at 68°C
4–24 hours at 42°C
3.
Carefully remove the hybridization solution by cutting one corner. Remove hybridized
membrane from the plastic bag.
Note: Once hybridization has begun, do not let the membrane dry.
Washes
1.
Agitate the solutions when washing membranes.
For DNA or RNA Bound to
For DNA Bound to
For RNA Bound to
Zeta-Probe Membrane
Nitrocellulose
Nitrocellulose
A.
Wash 2 times for 30–60
A.
Rinse in:
A. Wash 4 times at room
minutes at 65°C in:
2x SSC, 0.5% SDS
temperature for 5 minutes
1 mM EDTA
in 2x SSC, 0.1% SDS
40 mM NaHPO
4
, pH 7.2
5% SDS
B.
Wash 2 times for 30–60
B.
Wash at room
B. Wash 2 times at 50°C in:
minutes at 65°C in:
temperature for 5 minutes
0.1x SSC, 0.1% SDS
1 mM EDTA
in: 2x SSC, 0.5% SDS
40 mM NaHPO
4
, pH 7.2
1% SDS
C. Wash at 68°C 2 times
for 1 hour each in:
0.1x SSC, 0.5% SDS
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