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

Pre-Hybridisation

Hybridisation

Thermo Scientific
1. Once the membrane is in place in the bottle the SSPE (SSC) can be
discarded and replaced with pre-hybridisation fluid.
This is easily done by removing the cap, pouring off the SSPE (SSC),
then pouring in the pre-hybridisation fluid. All the solutions used
during hybridisation should be pre-warmed before use. 0.34-0.51 fl.
oz. (10-20 ml) solution is recommended for medium bottles and
0.17-0.34 fl. oz. (5-10 ml) for short bottles. Higher volumes will be
required if there is more than one membrane in the bottle, e.g.
0.51-0.85 fl. oz. (15-25 ml).
2. Replace the cap on the bottle and insert into the rotisserie.
1. Denature the purified probe by boiling for five minutes, then store on
ice. If the volume to be used for hybridisation is substantially different
than 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 pre-
hybridisation, simply pipette the probe into the pre-hybridisation 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 solution outside the bottle. Pour
off the pre-hybridisation solution 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.
Section 4
Methodology of Hybridisation
Shake 'n Stack 4-3