23
INSTALLING THE HYBRIDISATION TUBES
1.
Pre-heat the oven to the required temperature.
2.
Insert the tubes or tube holder into the
Hybridisation Oven
ensuring that they rotate freely and the
end caps
engage on the drive rollers on the right had side of the chamber.
3.
Close the door.
4.
Turn the speed control knob until you reach the required speed.
BASIC PROTOCOL FOR SOUTHERN HYBRIDISATIONS
Insertion of filters
Place a wet or moist filter, DNA side facing inwards, into a hybridisation tube and roll the tube, allowing
the filter 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 separation membranes between
filters, part number FMEM2020.
Pre-hybridisation
Add 5 or 10ml of hybridisation fluid, according to the number of filters and the size of tube, so that the
filters are covered. Allow to pre-hybridise for the required period.
Hybridisation
Drain off the pre-hybridisation fluid by removing the spindle cap from the fill and drain hole in the blue
end cap and inverting the tube. Add the hybridisation liquid together with the radioactive probe using a
syringe and needle through the fill and drain hole and replace the spindle cap. The minimum volume
for a single filter is 5ml (when using large tubes). Allow to hybridise for the required period.
Washes
Drain off the hybridisation fluid through the fill and drain hole, collecting the probe in a suitable
container for re-use or disposal. Washing depends on the stringency required and usually involves a
series of washes with SSC buffer and SDS of decreasing concentration until background radiation has
been removed.
Notes:
•
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.
WARNING
THE UNIT WORKS UP TO 80°C. This temperature can cause burns. Antylia Scientific Ltd.
takes great efforts to protect operators from hazards, but the following points are brought
to your attention:
1. Use care and wear protective gloves to remove hybridisation tubes from the cabinet if the unit is at
high temperature. If the protocol allows, it is better to switch off the unit and open the door to allow
the unit to cool to below 40°C before attempting to remove the tubes.
2. The coloured end cap of the 80mm large tube and the 44mm small tube has two holes. Take care
not to spill radioactive probe through these holes. One hole allows the air to expand when the tube
is heated. The other is the fill and drain hole which is revealed when the spindle cap is removed.
