Basic Protocol For Southern Hybridisations; When The Protocol Is Finished - Techne HB-1D Manual Del Usuario

H B - 1 D O P E R A T O R ' S M A N U A L

BASIC PROTOCOL FOR SOUTHERN HYBRIDISATIONS *

INSERTION OF FILTERS
Place a wet or moist filter, DNA side facing inwards, into a hybridisation tube and
allow it to adhere to the glass. Large trapped air bubbles should be smoothed out
with a glass pipette; smaller air bubbles are acceptable and do not interfere with
hybridisation so long as the surface of the filter is covered with hybridisation fluid.
Several filters may be placed in one tube; there is a slight loss of signal, but no
increase in background in the lower filters. To improve the signal we recommend
the use of seperation membranes, part number FMEM2020.
PRE-HYBRIDISATION
Add 5 or 10ml of fluid, according to the number of filters and the size of tube, so
that the filters are covered. Allow to pre-hybridise for usual period.
HYBRIDISATION
Drain off the pre-hybridisation fluid by inverting the tube. Add hybridisation liquid
with radioactive probe added by using a syringe and needle through the hole in the
blue end-cap. The minimum volume for a single filter is 5ml.
WASHES
Washing depends on the stringency required.
One method is to wash genomic blots in the following fashion:
1) 2 x SSC, 50ml for 10 minutes
2) 2 x SSC, 50ml for 10 minutes
3) 0.1 x SSC, 0.1% SDS x 50ml for 10 minutes
This gives very clean backgrounds.
NOTE
1)
2)
3)
* Courtesy of: Dr M J S Dyer.

When the Protocol is Finished

When the protocol has finished switch off the unit. Open the door and remove
the tubes, again being careful to keep the coloured end-cap uppermost. Remember
the tubes may be hot. Either wear gloves or, if the protocol permits, allow the tubes to
cool before removal.
20
All radioactive waste can be collected for disposal; none need be
disposed of down the sink.
Radioactive probes can be collected and reused several times.
As the protocols for this unit usually involve the use of radioactive
materials the appropriate manufacturer's safety precautions must be
taken into consideration.