Hybridization and Washing
Peter Hauser (last update 25.11.2002)
according to http://cmgm.stanford.edu/pbrown/protocols/index.html
Procedures
Hybridization:
- 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
Washing:
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