36 Microarrays and DNA-Chips in the Analysis of Genetic Aberrations
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in the size and sequence (i.e. secondary structure) of both the probe and
target. We strongly advise to check the sample and reference DNAs for
purity before labeling, and afterwards for efficiency oflabel incorporation.
Pre-hybridization
Arrays were prehybridized and washed as follows:
1. 2 x SSC/O.05% SDSIO.2%BSA at 42 DC for 20 min.
2. 1 x SSC at room temperature for 5 min, repeat once.
3. 0.2 x SSC for 5 min, repeat once.
4. After the final wash, slides were spin dried in a centrifuge at 2000 rpm
for 1 min.
Hybridization
Combine in a 1.5 ml microcentrifuge tube:
Hybridization solution
- 20 III 100% formamide
- 5 III 20 x SSC
- 1 III 10 IlgIll1 poly A
- 2 III 10 Ilgllli Human Cot-l DNA
- 1 III 5% SDS
- 2-5 III Cy3 test DNA (4 Ilg)
- 2-5 III Cy5 reference DNA (4 Ilg)
add DI water to a final volume of 50 III
1. Vortex the hybridization solution, then microcentrifuge at 12,000 rpm
for 5 s.
2. Incubate in a 95 DC water bath for 5 min in order to denature all DNAs.
3. Microcentrifuge at 12,000 rpm for 1 min at room temperature.
4. Add 5 III of 2% BSA to the hybridization solution and mix well.