User Manual

Washes

1. At the completion of hybridization, remove the membranes from their hybridization bags into

2x SSC. Rinse briefly, then wash them sequentially with agitation for 15 minutes at room

temperature in the following solutions:

a. 2x SSC/0.1% SDS

b. 0.5x SSC/0.1% SDS

c. 0.1x SSC/0.1% SDS

2. For DNA bound to nitrocellulose membranes, it may be necessary to include an RNase

treatment in the wash. Membranes are treated with 20 µg/ml RNase for 30 minutes at 37°C

in 2x SSC. (Santzen et al. 1986)

3. After washing, the blotted membranes are ready for autoradiography. If no further cycles of

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

reprobing, do not allow the membrane to dry between hybridizations. Expose moist membranes

between plastic wrap or enclosed in a sealable plastic bag. Do not allow a wet membrane to

come in contact with the film, because a wet Zeta-Probe membrane will stick to the film, and

moisture on the film can cause artifacts.

Note: To increase the rate of hybridization, include 10% dextran sulfate (final concentration)

in the hybridization solution.(Maniatis 1982) Prewarm hybridization solution to 50°C.

Denature the probe and carrier as above. Special care must be taken to ensure uniform

mixing of the denatured probe with the hybridization solution, since the solution is quite

viscous at 50°C.

7.4 Probe Stripping and Rehybridization

If reprobing is desired, do not allow the membrane to dry between hybridizations. The

membrane should be stripped as soon as possible after autoradiography.

1. Wash two times, 20 minutes each, in a large volume of 0.1x SSC/0.5% SDS at 95°C.

2. Check membrane for removal of autoradiography patterns by overnight exposure.