Biochemical Techniques and Instrumentation (1+2)

Hybridization

1. Add the probe to the edge of the prehybridization solution without squirting directly onto the blot by pipetting and mix probe into solution so that it is uniformly spread over the blot.
2. Place the seal-a-meal bag on a glass plate and gently roll out all bubbles with a 5 ml disposable pipet. Seal the final side of the seal-a-meal bag 0.5 cm from the top of the blot.
3. Wash the seal-a-meal bag well with running water and place in a 42^oC water bath overnight.
4. In the morning remove the blot from the bag directly into 500 cc of 2X SSC/0.1% SDS. Wash with gentle shaking at room temperature for 10 minutes. Repeat the 2X SSC/0.1% SDS wash for an additional 10 minutes at room temperature with gentle shaking.
5. Rinse the blot with a small volume of 0.1X SSC/0.1% SDS at room temperature for 10 seconds and discard.
6. Add 500 cc of 0.1X SSC/0.1% SDS to the blot in a tupperware container. Seal the top and place at 65°C for 30 minutes. Repeat this 65°C wash for an additional 30 minutes.
7. Remove the blot onto clean sheet of Whatman paper and listen to it with a Geiger counter. It should not be hot.
8. Let the blot air dry until surface liquid is gone, but the blot is still damp.
9. Wrap the blot in saran wrap and tape to a clean sheet of Whatman paper which has been marked asymmetrically with dots of radioactive ink.
10. Autoradiogram using an intensifying screen at -70°C for 4-24 hours.