This protocol is an amalgam of protocols from Jon Pollack, Joe DeRisi, and the unattributed protocols on the Brown lab web page.
DNA preparation (Hoffman and Winston)
Innoculate 10 ml YPD with a fresh colony.
Grow to saturation
Spin down and resuspend in 500 microliters water. Transfer to Eppendorf tube.
Spin down. Decant supernatant and resuspend pellet in residual liquid.
Add 0.2 ml lysis buffer.
Add 0.2 ml phenol-chloroform-isoamyl alcohol (25:24:1).
Add 0.3 g acid-washed glass beads (425-600 microns).
Vortex 3-4 minutes.
Add 0.2 ml TE.
Spin 5 minutes.
Transfer aqueous layer to new tube.
Add 1 ml 100% ethanol. Invert to mix.
Spin for 2 minutes.
Resuspend pellet in 0.4 ml TE plus 20 micrograms RNaseA.
Incubate 5 minutes at 37 degrees C.
Add 10 microliters 4 M ammonium acetate and 1 ml 100% ethanol. Invert to mix.
Spin 2 minutes.
Resuspend in 50 microliters TE. 10 microliters contains roughly 2-4 micrograms DNA.
2% Triton X-100
100 mM sodium chloride
100 mM Tris pH 8
1 mM EDTA
labelingCut 1-2 microliters DNA with HaeIII or HinP1I (or any other 4-cutter) in 25 microliters for >1 hour.
2 minutes each, at room temperature.
20 ml 20X SSC
1.2 ml 10% SDS
Let coverslip float off in this wash. Let arrays sit in this solution until all coverslips are off, then start 2 minute agitation. Transfer each slide individually to wash 2 to minimize SDS carryover.
4 ml 20X SSC
1 ml 20X SSC
Spin dry at 500 RPM in a tabletop centrifuge. Scan.