Prehybridisation; Hybridisation - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Prehybridisation

1. Once the membrane is in place in the bottle the SSPE (SSC) can be discarded and
replaced with prehybridisation buffer.
This is easily done by removing the cap, pouring off the SSPE (SSC) and then
pouring in the prehybridisation buffer. All the solutions used during Hybridisation
should be pre-warmed before use. 10-20ml solution is recommended for medium
bottles and 5-10ml for small bottles. Higher volumes will be required if there is
more than one membrane in the bottle,
e.g. 15-25ml.
2. Replace the cap on the bottle and insert into the rotisserie.

Hybridisation

1. Denature the purified probe by boiling for 5 minutes, then store on ice. If the
volume to be used for Hybridisation is substantially different to that used previously
for Hybridisation in bags or boxes, ensure that the quantity of the probe is adjusted
accordingly to maintain the correct probe concentration. If this is not done, high
background may result.
2. Remove the bottle from the Hybridisation Oven and unscrew the cap.
3. If the same buffer is to be used for Hybridisation as for prehybridisation simply
pipette the probe into the prehybridisation fluid in the bottle. Take care to avoid
pipetting the probe directly on to the membrane as this will result in hot spots.
Alternatively, dilute the probe in pre-warmed Hybridisation buffer outside the bottle.
Pour off the prehybridisation buffer and replace with the probe solution.
4. Replace the cap and gently agitate the bottle to ensure an even distribution of the
probe in the Hybridisation solution.
5. Place the bottle back in the Hybridisation Oven, switch on the rotisserie and leave
it to hybridise for the required time period.
© Thermo Scientific, May 2003. Issue 7
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