UVP HybriCycler - Manual (Page 13)

HC-3000 HybriCycler Hybridization Oven 13
5. Denature the probe by placing the samples in the boiling water bath
and heating for 10 minutes. Immediately transfer the tube to ice for 5
minutes (to prevent renaturation). Add 5 mL of hybridization buffer to
the probe and transfer to the hybridization bottle containing the blot:
AVOID pouring the probe directly onto the blot.
6. Incubate in the UVP HybriCycler, Hybridizer, or Minidizer 6 to 8 hours
at 42 to 56 degrees.
Washing the blot
Tupperware container (sized to contain the blot)
0.1X SSC, 0.1% SDS (pre-warmed to 50 °C)
2X SSC, 0.1 % SDS (room temperature)
2X SSC (room temperature)
0.15X SSC, 0.1% SDS (pre-warmed to 50 °C)
Gloves
Filter Paper
Cardboard
Plastic wrap
Tape
Non radioactive probes
1. Wash blots in 2X SSC, 0.1% SDS for 3 minutes at room temperature
(repeat one)
2. Wash filter in 0.15X SSC, 0.1%SDS for 15 minutes at 50°C (repeat once)
3. Store blots in 2X SSC at room temperature
Radioactive probes
Additionally you will need:
X-Ray film holder
X-Ray film
Intensifying screen
Procedure
1. Remove blot from hybridization tube and transfer to Tupperware
container
2. Rinse briefly in 50°C 0.1X SSC, 0.1% SDS.
3. Remove this solution to radioactive waste and wash
4. Wash blot three more times in the same solution.
5. After the final wash, dry blot on filter paper for 10 minutes. This is a
good time to quickly pass your hand held radioisotope reader (beta or
gamma counter) over your blot to get a general idea as to the
exposure time you will need for the x-ray film. Hot blots are 20
minutes to 2 hours. Not so hot blots can be left overnight (8 hours).