Lance D. Miller

NIH-NCI-DCS-MSO

(adapted from J. DeRisi;

Pat Brown lab, Stanford)

 

Post-Processing Slides

 

We have found that we achieve best results by storing the printed arrays (in a sealed slide box at room temperature) for 5-7 days prior to post-processing. Once post-processed, the arrays are stored in a sealed container (i.e. vacuum chamber, parafilm-wrapped slide box, etc.), and the shelf-life has been measured out to at least 6 months for optimal performance.

 

A. Preparations

 

  1. After printing, gently number slides and mark array borders with a diamond-tipped scriber (VWR) and transfer slides from the print platter to 30-slot slide racks. Store in glass dishes.
  2. Fill a 2.5 L beaker with enough ddH2O to cover the slide rack. Put beaker on hot plate set at maximum heat. Boiling water will be used to denature the DNA arrays.
  3. Fill a clean glass dish with 350 ml 95% EtOH, and another with 350 ml distilled or deionized H2O.

 

B. UV Crosslinking

 

  1. Lay slides array-side-up inside a Stratalinker. Adjust the setting for 600 (x 100) microjoules.
  2. Make sure the “Energy” button is lit, and push start.
  3. Turn the Stratalinker off and transfer slides back to the 30-slot racks.

 

C. Succinic Anhydride Blocking

 

  1. Add 6.0 g of succinic anhydride to a 500 ml beaker with stir bar.
  2. Pour 335 ml 1-methyl-2-pyrrolidinone into the beaker and stir.
  3. Once the succinic anhydride has dissolved, add 15 ml of 1 M Boric Acid pH8.0 to the beaker and stir thoroughly 10-20 seconds.
  4. Pour this solution into a clean glass dish.
  5. Submerge a rack of arrays (dunk 7-10 times vigorously to prevent pluming!) and place on orbital shaker for 15 minutes.
  6. Remove rack and place in the glass dish containing 350 ml ddH2O. Rinse slides by plunging rack several times (~10 seconds).
  7. Your large beaker of water should be boiling by now. Turn off the heat, and as soon as the bubbling subsides, transfer the rack into the beaker for 2 minutes.
  8. Quickly remove the rack and place in the dish containing 350 ml 95% EtOH. Plunge several times (~10 seconds).
  9. Transfer to the centrifuge microplate carrier and spin for 3 minutes @ 60-100 xg. (Use a balance at this step; i.e. another 30-slot rack with blank slides.)
  10. Transfer slides to a clean plastic slide box for storage (no cork!!). Store tightly sealed or in a  vacuum chamber at room temperature.