PROBE LABELING

Note: limit exposure of Cye Dyes to light at all times (this can be achieved by working in a darkened area or by shielding the tubes with a cover such as aluminum foil)

1. Combine:
   (Setup 1 reaction tube for reference and 1 reaction tube for sample)
   • DNA (25-400 ng)
   • 5 µL of 5X random primers buffer (Final concentration: 5X Promega Klenow buffer and 7 µg/ µL random octamers)
   • Dilute to 17.0 µL total volume with distilled H₂O
2. Boil for 10 minutes at 100°C. Transfer immediately to ice for 1 minute.
3. Add 4 µL of 10X dNTP mix (2mM dATP, dGTP, dTTP, 1.2mM dCTP).
4. Add CyeDyes:
   • Add 2 µL (2 nmoles) of Cy-3 labeled dCTP to Sample DNA
   • Add 2 µL (2 nmoles) of Cy-5 labeled dCTP to Reference DNA (e.g., Novagen human genomic DNA)
5. Add 2.5 µL of Klenow (9 U/µL, Promega ) and mix.
6. Incubate at 37°C overnight* (~18 hours).
   
   *The overnight hybridization time can be adjusted between 14 to 24 hours without adversely affecting the labelling process to fit to a laboratory schedule.

SAMPLE CLEAN UP

(Combined probe clean-up and preparation for hybridization)

1. Using a Microcon YM-30 column:
   • Add 100 µL Cot-1 DNA (1µg/µL) to column. Do not touch membrane with pipette tip.
   • Pool the reactions (reference and sample) and add to column.
   • Place column in provided tube and spin at 13000 g for 10 minutes.
   • Add 200 µL distilled H₂0 to membrane and repeat spin to wash.
   • Discard tube and add 45 µL hybridization solution: (Roche DIG Easy)
   Optional: add 5µL sheared herring sperm DNA (10µg/µL unit, Promega)
   
   • Invert Microcon, place in a new labeled tube, and spin at 3000 g for 3 minutes.

CALCULATION OF INCORPORATIONS

1. Remove 1.5 µL and measure Fluorophore incorporation using NanoDrop Spectrophotometer.  Using your hybridization buffer as a blank (DIG Easy) follow the directions on the screen. (You can recover the sample from the pedestal, if necessary, after measurement.)
   
   (Note: Incorporations below 3.0 pmol/µL in either channel have shown variable results.)
   
   
2. Denature at 85°C for 10 minutes.
3. Place probe at 45°C for 30 minutes to 1 hour (allows Cot-1 DNA annealing.)

ARRAY HYBRIDIZATION

1. Place 44 µL probe solution onto the coverslip (22 mm x 60 mm) (Fisher Scientific). If available, place the coverslip on a slide warmer or heat block pre-warmed to 45°C to maintain temperature.
2. Align slide over coverslip and probe solution, lower one edge of the slide allowing contact with the hybridization solution. Continue to lower until the coverslip is attached to the slide with surface tension. Invert slide.
3. Place the slide into hybridization cassette (Telechem), pre-warmed to 45°C, and add 10 µL of water in the lower groove (to control humidity during hybridization).
4. Seal cassette and incubate for 36 - 40 hours at 45°C.

ARRAY WASHING

Note: All wash solutions are at pH 7.0

Removal of the slide from the hybridization cassette is critical. DIG Easy quickly crystallizes at room temperature. The slide should be immediately immersed and the cover slip removed in the wash solution.

1. Add approximately 60 mL of 0.1XSSC, 0.1%SDS (pH 7.0, 45°C) wash solution to a Coplin jar prior to opening the hybridization chamber.
2. Open chamber, add slide to wash solution and remove coverslip (it should slide off).
3. Wash the slide 3 times in 0.1XSSC + 0.1%SDS at 45°C for 1 minute each with agitation.
4. Rinse the slide 3 times in fresh 0.1XSSC*. There should be no residual bubbles from the SDS wash visible.
   
   *The slides can remain in the final wash solution until ready to centrifuge and scan (~15 minutes). 
   
   
5. Centrifuge the slide in a 50 mL Falcon tubes at 700 g for 3 minutes.
6. Immediately scan the slides (signal intensities will diminish over time.)