Hybridization and Washing

Peter Hauser (last update 25.11.2002)

according to http://cmgm.stanford.edu/pbrown/protocols/index.html



  • Adjust sample volume to 32 µ with nuclease free H2O
  • Add 6.8 µl 20x SSC and 1.2 µl 10% SDS to end volume of 40µl
  • Boil samples at 95°C for 1 minute
  • Incubate at 37°C for 20 minutes
  • Place 30 µl 3xSSC under the slide
  • Pipette the sample on the microarray, be sure not to touch the array with pipette tip
  • Place the cover-slip carefully on the array
  • Avoid air bubbles and don´t scratch the surface!
  • Close hybridisation chamber and place the chamber in a 65°C waterbath for 14-18 hours


Buffer 1: 2x SSC + 0.1% SDS 65° C
Buffer 2: 1x SSC RT
Buffer 3: 0.1X SSC RT
  • Use 3 slide chambers for the washing procedures. Use separate slide racks for wash 1 and 2, to avoid transferring too much SDS
  • Transfer the microarrays upside-down into wash chamber 1 and agitate for 5 minutes in wash buffer 1 on a orbital shaker
  • The cover-slip will fall off during this step!
  • Transfer slides in a new rack and agitate in wash buffer 2 for 5 minutes
  • Transfer slides to wash buffer 3 and shake for 5 minutes
  • Spin the slides in the slide-rack for 3 minutes with 200g at room temperatu