Hybridization of Arrays

Friday, November 21, 2003

Description
Hybridization of Arrays

Procedure
Directions for fluorescent DNA probes:
1. Final probe volume should be 12-15uL, at 4X SSC, containing competitor DNA etc. as required
(e.g., polydA, CoT1 DNA for a human cDNA array)
2. Set up array in hybridization chamber.
Place 10 uL 3X SSC on edge of slide to provide humidity.
3. Add 0.3 uL 10% SDS to probe.
4. Boil probe for 2 min. Set aside several cover slips for next step.
5. Pipet probe onto array, avoiding bubbles.
Using forceps to help, immediately lay down 22mm x 22mm cover slip over array, avoiding bubbles.
It is worth practicing this step several times with 10 uL water and a blank microscope slide.
6. Close hybridization chamber and submerge in 65C water bath.
7. Hybridize 4-24 hr.
8. Prepare wash solutions #1 (1X SSC / 0.03% SDS), #2 (0.2X SSC), and #3 (0.05X SSC):
WASH 1 WASH 2 WASH 3
dH2O 190 198 200
20X SSC 10 2 0.5
10% SDS 0.6 -- --
Final vol.: 200 mL 200 mL 200 mL
9. Pour wash solutions in slide chambers, and place slide racks in washes #1 & #2.
10. Disassemble hybridization chamber and quickly submerge array in wash #1.
(If the array is exposed to air while the cover slip starts to fall off, you may see high background fluorescent signal on the side of the array.)
11. Let the array sit in wash #1 until the cover slip slides off.
Gently plunge slide rack up and down several times to wash the array;
be sure not to scratch the array with the loose cover slip.
9. Manually transfer array to slide rack in wash #2 and rinse 2nd time.
10. Move slide rack to wash #3 and rinse 3rd time. It is critical to remove all SDS.
11. Centrifuge slides on microtiter plate carriers (place paper towels below rack to absorb liquid) for 5 min. @ 500 rpm.
12. Scan array immediately. Notes:
- Be aware of Cy5 bleaching during scanning.

Recipes
Materials Qty Order info
100C heat block 1
65C water bath 1
Hybridization chamber 1 per array Custom made by Monterey Industries or purchased at Corning Costar (#2551)
Cover slip, 22 mm X 22 mm 1 per array
3X SSC 10 uL per array
10% SDS ~1 mL
Fine-tip forceps 1
Slide rack 2 TBA <= Each rack holds 8 slides
Slide chamber 3 TBA <= Each chamber holds 200 mL
ddH2O ~600 mL
20X SSC ~13 mL

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Hybridization of Arrays