Post-Processing of Oligo Arrays on PolyLysine coated slides

Kate Rubins

DRAFT VERSION 01.25.06

USE THIS FOR POLY-LYSINE SLIDES ONLY. DO NOT USE ON CORNING ULTRA GAPS, SCHOTTS AMINO SILANE OR OTHER NON POLY LYSINE SURFACES.

Rehydration/Shampoo

  1. Mark boundaries of array on back of slide using diamond scriber. Prop slide between two tip boxes with lamp overhead and etch corners of array. Array will become invisible after post-processing.
  2. Expose slides to 65mJ of UV irradiation (Place slides inside, face up, on a paper towel. Set the Stratalinker to Energy and type in 650).
  3. Make shampoo solution:
    66mL of filtered 20X SSC
    8mL of 10% SDS
    332mL of distilled H20
  4. Preheat shampoo solution in microwave (heat for 2-3 minutes and check) to just above 60°C
  5. Put solution in Hybex Chamber (set at 60°C) and let solution equilibrate with Hybex lid closed for at least 10 minutes. Check solution temperature with a thermomether, solution temperature should be 50°C
  6. Set up a bath of 400mL H2O at Room Temperature next to the Hybex
  7. Set up a bath of 400mL 95% Ethanol at Room Temperature next to the Hybex
  8. Dunk slides into Shampoo Solution at 50°C, plunge for 15 seconds, close the Hybex cover and incubate for 5 min. Check the temperature during the incubation.
  9. Transfer to 400mL H2O for a few seconds to rinse
  10. Transfer to 400mL 95% EtOH for a few seconds.
  11. Spin slides at 500 RPM, 5 min. Room Temperature. Quickly and evenly transfer the slides to the centrifuge to prevent streaking.
  12. Place arrays in metal slide rack, every other slot. Be sure the rack is bent slightly inwards in the middle, or else the slides may run into each other while shaking.

Succinic Anhydride Blocking

  1. Measure out 15ml sodium borate in a 50 ml conical tube.
  2. Measure out 5.5g succinic anhydride into a conical tube. (Use fresh stock, no older than 3 months.)
  3. Prepare shaker in the fume hood. Put 500mL glass chamber (with metal top) on shaker.
  4. Place a 500mL beaker with a stir bar on a stirring block in the hood.
  5. Prepare a second 500mL glass chamber with 95% EtOH.
  6. Prepare a third 500mL glass chamber with Room Temperature H20.
  7. Dissolve 5.5 g succinic anhydride in approx. 325-350 mL 1-methyl-2-pyrrolidinone (use fresh stock no older than one month). Rapid addition of reagent is crucial.
    1. Immediately after the last flake of the succinic anhydride dissolves, add the 15 mL sodium borate.
    2. Immediately after sodium borate solution mixes in, pour solution into empty slide chamber.
    3. Plunge slide rack rapidly and evenly in solution. Vigorously shake up and down for a few seconds, making sure slides never leave solution.
    4. Mix on orbital shaker 15-20 min.
  8. Gently plunge slide rack in Room Temperature H20 for 60 seconds.
  9. Plunge slide rack 5X in 95% ethanol.
  10. Centrifuge slides and rack for 5 min. @ 500 rpm. Load slides quickly and evenly onto the carriers to avoid streaking.
  11. Use arrays immediately or store in slide box.