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RPN2510EUM Chapter 7 Rev C 06/2007 11
4. Prehybridize the membrane for the specified length of time at a rotisserie speed
of 4 rpm.
5. Following prehybridization, turn off the rotisserie, remove the rotisserie from the
oven. Add the labeled probe to the buffer, either by removing an aliquot of the
buffer, adding the probe and returning the aliquot to the bottle; or by adding the
probe directly into the bottle, avoiding the membrane.
6. Hybridize for the specified length of time at a rotisserie speed of 4 rpm.
7.3. Membrane washing procedures
Membranes can either be removed from the hybridization bottles and washed in a
box on the platform shaker (this is a more efficient procedure), or the washing
procedure may be carried out in the bottles. If the platform shaker is used, follow
the standard washing procedure mentioned in the appropriate protocol booklet.
If bottles are to be used it is necessary to modify the standard washing procedure.
Outlined in this section are the optimized washing protocols for radioactive
hybridizations and non-radioactive based hybridizations.
7.3.1. Radioactive Hybridizations
1. Carefully drain off the hybridization buffer, rinse the bottle and the membrane
thoroughly with 2 × SSC and discard.
2. Perform the following stringency washes in large volumes (100 ml minimum) of
the following solutions, at a rotisserie speed of 8 rpm:
2 × 10 minutes with 2 × SSC, 0.1% SDS at 65°C
1 × 15 minutes with 1 × SSC, 0.1% SDS at 65°C
2 × 10 minutes with 0.1 × SSC, 0.1% SDS at 65°C
(More washes over the same time period for each stringency condition can
improve background).
3. Remove the membrane from the bottle, drain off excess stringency wash, wrap
in SaranWrap™ and autoradiograph.
7.3.2. AlkPhos Direct Hybridizations
4. Drain off the hybridization buffer, rinse the bottle and the membrane thoroughly
with primary wash buffer and discard.
5. Perform the following stringency washes in large volumes (100 ml minimum) of
the following solutions, at a rotisserie speed of 8 rpm:
3 × 10 minutes with primary wash buffer solution at 55°C
3 × 5 minutes with secondary wash buffer at room temperature
6. Remove the membrane from the bottle and detect using the standard
procedures outlined in the protocol booklet.
NOTE: This last step is a high stringency
wash and should be omitted if related
sequences are to be probed.
NOTE: The room temperature washes
can be achieved by switching off
the oven and leaving the door open
whilst performing the washes or by
allowing the oven to cool down to room
temperature before performing the
final washes.