1. Rinse the blots in 50 ml of 2x SSPE for 5 minutes. Drain and discard the solution.

2. Pre-warm the hybridization oven to 65^oC. Warm the hybridization solution to 65^oC for about 10 min prior to use. Pre-hybridize the Panorama gene array in 5.0 ml Hybridization Solution for at least 1 hr at 65^oC, using roller bottles at 6 r.p.m. Note: Chemicals may come out of the hybridization solution at storage of 4^oC. Pre-warming to 65^oC ensures that all components are dissolved. Add Salmon sperm DNA (final concentration of 100mg/ml) to pre-hyb and hybridization solutions immediately prior to use.

3. Denature the 400-600 ml cDNA generated probe (90-95^oC for 10 min) and add to 3 mls hybridization solution.

4. Decant and discard the pre-hybridization solution from the array. Add the denatured, labeled cDNA in the hybridization solution to the array roller bottle (30cm x 3.5cm)

5. Hybridize overnight (12-18 hrs) at 65^oC.

6. After hybridization, pre-warm the Wash Solution to 65^oC.

7. Decant hybridization solution into P³³ liquid waste.

8. Add 50 mls Wash Solution to the roller bottle. Wash the array by inverting roller bottle at room temperature on the inverting shaker for 5 minutes. Alternatively, hand roll the bottles for 5 minutes behind a radiation shield. Decant and discard the wash solution.

9. Repeat step 8 two more times

10. Add 80 ml Wash Solution & incubate/wash in hybridization oven at 65^oC for 30 min at 6 r.p.m.

11. Repeat step 10 two more times.

12. Decant Wash Solution and use forceps to gently pull out the array (allowing the solution to run down the inside of the hybridization tube).

13. Store the membrane. There are possible methods for doing this.

• Roll up the moist membrane and place it into a clear sheet protector.
• By rolling up the membrane it is much more manageable to place it into the clear sheet protector.
• Once it is inside the sheet protector it can be easily unrolled.
• Take a glass pipet and roll it over the membrane inside the sheet protector to push out bubbles thus ensuring the membrane is snuggly surface tension retained to the sheet protector.
• Quickly seal around the membrane using a lab bag sealer (low setting or you will melt through the plastic) to prevent any air bubbles from entering.

• Use Saran wrap, however this is not an airtight method and the membrane will eventually dry out.
• In addition residual SDS left from the last membrane wash step can damage the phosphoscreens!
• It is also important when using Saran wrap to make sure that you don't crease the membrane or wrinkle the Saran Warp when folding the Saran Wrap around the array.
• Wrinkles will prevent the membrane from sitting flat against the phosphoscreen resulting in a blurry image!
  
  

Hybridization Solution (store at 4^oC) (Makes 100 mL)

Wash Solution (Makes 1 L)

20 X SSPE (3.6 M NaCl, 0.2M sodium phosphate, 20 mM EDTA) (Makes 1 L. Store at 4^oC)

in 800 ml nanopure H₂O
Adjust to pH 7.7 with NaOH pellets. Adjust to 1 L with nanopure water. Autoclave & store at 4^oC.

50X Denhardt’s Reagent (1% Ficoll, 1% PVP, 1%BSA):

in nanopure dH₂O to 500ml, filter sterilize and store (-20^oC)