Spotted Microarray Hybridization and Scan
Note: for single spotted arrays halve
Mix two color samples (for genomic DNA, Normally 0.5ug), add 2ml
Block Mix (10mg/ml salmon sperm DNA (BRL) + 4mg/ml yeast tRNA)
Use Speed Vec to dry the sample to a small volume, normally less
than 5ml, do NOT overdry.
Add 50ml Sigma ArrayHybTM Hybridization Buffer
Heat mixture to 100°C 5 minutes, mix by pipetting up and down
Spin for 5 minutes at 14,000 RPM to pellet any particulate material
Place the Microarray(s) in a hybridization chamber (TeleChem or
GeneMachines)(To prevent arrays from drying, seal array in hybridization
chamber with Whatmann paper moistened with 100 ml of H2O.)
Carefully transfer the probe solution to a new tube avoiding any
Carefully pipette the hybridization mix onto the array (between
marks etched on back of array)
Overlay with a clean glass cover slip being careful to avoid bubbles
under the cover slip
Seal chamber and place in 60°C water bath. Incubate 18-20 hours
Dry the exterior of hybridization chamber
Carefully open hybridization chamber to avoid H2O entering
Remove slide from the chamber and quickly place the slide, array
side down, in a container filled with 0.2X SSC, 0.03% SDS and a slide
holder. Position the slide upside down and at a slight angle so the
coverslip falls away from the slide. Keep slide in this position until
the coverslip has fallen off. When cover slip falls off, transfer
the slide to the other wash chamber containing wash buffer 1 and incubate
for 5min at room temperature with gentle mixing on an orbital shaker
Transfer ONLY the slide to a container with 0.2X SSC (filtered)
and a slide holder. Incubate for 5min at room temperature with gentle
mixing on an orbital shaker (speed 3-4).
Transfer the slide holder with the slide to fresh 0.2X SSC (filtered)
and repeat wash 5min.
Transfer the slide holder with the slide to 0.05X SSC (filtered)
and wash for 45 sec.
Spin in slide holder seated in a Plexiglass carrier on microtiter
plate carrier for 5 minutes at 500 RPM to dry slide.
Currently we use Packard Science SA5000 scanner to scan our microarrays.
to obtain good signal and low signal/noise, different PMT setting might
be used. However, to compare probe quality and hybridization quality,
it is suggested to scan all slide at the same power and PMT setting.
Now we use the setting:
- For Cy3, which normally is the RNA channel, Power 85, PMT 75.
- For Cy5, which normally is the genomic DNA channel, Power 85, PMT
||Cat # AHC-1
||Cat # A-7718
Please let me know of any problems, suggestions or updates to this