User Manual

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

Zeta-Probe membrane from the plastic bag.

Note: Once hybridizationhas 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 two 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

, pH 7.2

5% SDS

B. Wash two times for 30–60 B. Wash at room B. Wash two times at

minutes at 65°C in: temperature for 50°C in:

1 mM EDTA, 5 minutes in: 2x SSC, O.5% SDS

0.1x SSC, 0.1% SDS, 1 mM EDTA

40 mM NaHPO

, pH 7.2,

1% SDS

2. After washing, the blotted membrane is ready for autoradiography. If no further cycles of

hybridization are to be done on the membrane, the membrane can be dried. When

reprobing, do not allow the membrane to dry between hybridizations. Make the

autoradiographic exposure with the moist membrane wrapped in plastic wrap. Do not

allow the wet membrane to come in contact with the film, because a wet Zeta-Probe

membrane will stick to the film, and any moisture on the film will cause artifacts (black

spots).