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RPN2510EUM Chapter 6 Rev C 06/2007 9
6. Hybridization using the platform
shaker
The Hybridization Oven/Shaker is compatible with the hybridization technologies
available from GE Healthcare. These include radioactive hybridizations using
Rapid hyb buffer and the range of non-radioactive systems for the labelling and
detection of proteins and nucleic acids (see appendix 1).
When using the platform shaker the hybridization and washing conditions
recommended in the appropriate GE Healthcare literature should
be used. The following protocol therefore provides a guideline. For specific
hybridization times and temperatures, refer to the relevant protocol booklet.
1. Prepare blots as recommended in the appropriate Hybond protocol booklet.
2. Set the oven temperature as described in section 5.1.
3. Place the membrane in a suitable box (or bag) and cover with sufficient
prehybridization buffer to ensure that the entire surface of the membrane is
covered. Recommended volume: surface area ratio is given in most protocol
booklets. Seal the box (or bag) securely.
4. Place the box (or bag) on the platform, set the oscillation speed to 30 strokes/
minute as described in section 5.3. Prehybridize for the required length of time.
5. Remove the box (or bag) from the oven and carefully add the labeled single
stranded probe (denaturation may be required post labelling refer to the
appropriate protocol booklet) to the prehybridization buffer.
6. Reseal the box (or bag) securely, replace it on the platform and hybridize for the
required length of time.
7. Remove the membranes and place in a clean box containing the first stringency
wash solution.
8. Increase the oscillation speed of the platform to 60 strokes/minute. Carry out
the recommended washing protocol at the appropriate temperature and for the
appropriate times.
NOTE: Do not pipette the probe solution
directly on to the membrane as this
may cause localized background.