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UWE R. MOLLER et a1.
5. Apply entire hybridization mixture (final volume 50 JlI) onto the array
and then spread over the entire surface using a 24 mm x 50 mm cover
slip.
6. Incubate chip(s) at 42 DC in the dark for overnight (15-18 h).
Post-hybridization processing
7. Wash chip(s) in a series of 8 Coplin jars as follows:
- 1 wash in 2 x SSC/O.05% SDS at 42 DC for 1 min to remove the cover
slip
- 2 washes in 2 x SSC/0.05% SDS at 42 DC for 5 min
- 2 washes in 1 x SSC for 5 min each at RT
- 3 washes in 0.2 x SSC for 2 min each at RT
8. Remove chips from last wash, and dry by centrifugation at 2000 rpm
for 1 min.
Note: Adequate washing is vital to assay performance. When transferring chips from one bath to the next agitate the biochip with an up
and down and back and forth motion to ensure proper washing.
Note: Do not allow chips to dry between washes!
Note: Any fingerprints, dust or lint on the chip will affect the image.
Thus, it is critical to remove any dust with a clean source of compressed air.
Image acquisition and analysis
The GenePix 4000A (or 4000B) Array Scanner was used to obtain the Cy3/
Cy5 fluorescence images using a PMT setting of 750-950 volts. All images
were analyzed using the GenePix Pro 3.0 analysis software (Axon Instruments, Inc., Foster City, CA). For determination of copy number changes
we make the assumption that most genes are represented by 2 copies. The
background corrected test/reference fluorescent intensity ratios are then
devided by the median ratio for all spots to obtain the normalized ratio of
1. We typically use self/self hybridizations to test the performance of our
assay system with model arrays and find that the normalized ratios have a
CV of approximately 28%.
UWE R. MOLLER et a1.
5. Apply entire hybridization mixture (final volume 50 JlI) onto the array
and then spread over the entire surface using a 24 mm x 50 mm cover
slip.
6. Incubate chip(s) at 42 DC in the dark for overnight (15-18 h).
Post-hybridization processing
7. Wash chip(s) in a series of 8 Coplin jars as follows:
- 1 wash in 2 x SSC/O.05% SDS at 42 DC for 1 min to remove the cover
slip
- 2 washes in 2 x SSC/0.05% SDS at 42 DC for 5 min
- 2 washes in 1 x SSC for 5 min each at RT
- 3 washes in 0.2 x SSC for 2 min each at RT
8. Remove chips from last wash, and dry by centrifugation at 2000 rpm
for 1 min.
Note: Adequate washing is vital to assay performance. When transferring chips from one bath to the next agitate the biochip with an up
and down and back and forth motion to ensure proper washing.
Note: Do not allow chips to dry between washes!
Note: Any fingerprints, dust or lint on the chip will affect the image.
Thus, it is critical to remove any dust with a clean source of compressed air.
Image acquisition and analysis
The GenePix 4000A (or 4000B) Array Scanner was used to obtain the Cy3/
Cy5 fluorescence images using a PMT setting of 750-950 volts. All images
were analyzed using the GenePix Pro 3.0 analysis software (Axon Instruments, Inc., Foster City, CA). For determination of copy number changes
we make the assumption that most genes are represented by 2 copies. The
background corrected test/reference fluorescent intensity ratios are then
devided by the median ratio for all spots to obtain the normalized ratio of
1. We typically use self/self hybridizations to test the performance of our
assay system with model arrays and find that the normalized ratios have a
CV of approximately 28%.