IMMOBILIZATION OF DNA ON SIGMA SILANE SLIDES

(Modified from Sigma Technical Bulletin MB-745 November 1999-Product No: A7718 ArrayHyb Buffer; this version from Betsy Pierson)

1. Mark the boundaries of the array on each slide using a diamond scriber (array will become invisible after post-processing). Be careful not to press so hard slides break.

2. Hold the slide (face down) over a 42 °C water bath for 5-10 seconds to the rehydrate spots. Note with high density printing (center to center spacing 200 um or less), do not exceed 10 seconds rehydration time or spots will run together. Lower density printings can be rehydrated up to 1 minute.

3. Snap-dry each slides (DNA side up) on a 70-80 °C inverted heat block for 3-10 seconds until the humidity is gone.

4. Place arrays in a slide rack and UV crosslink DNA to glass with 65 mJ of 254 nm UV light. (We use a Sigma Humidity Chamber {Sigma H6644} to hold the slides. We use a Fisher Scientific FB UVXL-1000 UV Cross Linker set to 650 x 100 uJ/cm2 for crosslinking DNA).

5. Incubate slides for ~ 2 minutes in 1% SDS on orbital shaker. This step removes the unbound nucleic acids from the arrays and prevents them from binding elsewhere. (We use Sigma staining chambers and racks {Sigma S6141} for all rinses. Make sure the volume in chamber completely covers slides while shaking).

6. Gently plunge the slide rack in 95°C water (just stopped boiling) for 2 min. We put the Sigma staining rack with slides directly into the water bath.

7. Rinse slides by plunging rapidly 10-20 times in a 100% ethanol bath (use a clean staining chamber and transfer slides to ethanol still in same staining rack).

8. Quickly transfer slides to 50 ml disposable centrifuge tubes and spin slides at 50-100 x g for 2-5 min. This step must be done quickly and carefully to avoid streaking and smearing arrays.

9. Use arrays immediately or store in a slide box (room temperature, with desiccant).