array CGH protocol 1 

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.
Dry pellet.
Resuspend in 50 microliters TE. 10 microliters contains roughly 2-4 micrograms DNA.

lysis buffer
2% Triton X-100
1% SDS
100 mM sodium chloride
100 mM Tris pH 8
1 mM EDTA


labeling

Cut 1-2 microliters DNA with HaeIII or HinP1I (or any other 4-cutter) in 25 microliters for >1 hour.
Heat inactivate enzyme.
Clean up with a spin cartridge like the Edge Biosystems Gel Filtration Cartridge or a Qiagen PCR cleanup kit.
Add 20 microliters 2.5X random primer reaction buffer (125 mM Tris pH 6.8, 12.5 mM MgCl2, 25 mM beta-mercaptoethanol, 750 ng/microliter random nonamers).
Boil 5 minutes. Ice.
Add 5 microliters 10X dNTP mix (1.2 mM each dATP, dGTP, dCTP, and 0.6 mM dTTP).
Add 3 microliters Cy-dUTP (1 mM stock) appropriately.
Add 1 microliter klenow.
Incubate 37 degrees C 1-2 hours.
Stop with 5 microliters 0.5 M EDTA pH 8.
Add 450 microliters TE pH 7.4 and add to microcon 30 concentrator.
Spin 8 minutes at 10000 rpm in microcentrifuge.
Invert column in a new tube and spin 1 minute to collect probe.
Combine Cy3 and Cy3 reactions. Add 10 micrograms yeast tRNA. Add TE to 500 microliters.
Concentrate in microcon to a volume of 24 microliters (or whatever's appropriate for the size array you have.)
Collect probe. Adjust volume to 24 microliters with TE. Add 5.1 microliters 20X SSC and 0.9 microliters 10% SDS.
Boil 2 minutes. Cool briefly. Apply to microarray, apply coverslip, place in hybe chamber, and hybridize overnight at 65 degrees C. Put 15 microliters 3X SSC in spots around edge of array for humidity.

washing

2 minutes each, at room temperature.

wash 1
400 ml
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.

wash 2
400 ml
4 ml 20X SSC

wash 3
400 ml
1 ml 20X SSC

Spin dry at 500 RPM in a tabletop centrifuge. Scan.