Analysis of Staphylococcus aureus Gene Expression During PMN Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 112

fragmented cDNA. Bring sample up to 100 μL with nuclease-free water ( see
Note 4 ).
2. Incubate the terminal labeling reaction at 37 °C for 60 min.
3. Stop the reaction by storing the sample at -80 °C until hybridization. The labeling
efficiency can be checked by performing a gel-shift assay. As a general rule, over
90% of the fragments should be labeled and thus shifted.
3.8. Hybridization of cDNA to Affymetrix GeneChips
The following protocol is designed for analysis of S. aureus transcripts on
Affymetrix 49 (standard) arrays.
1. Remove GeneChips from 4 °C storage. Allow chips to warm to room temper-
ature prior to hybridization (allow at least 60 min for GeneChips to equilibrate).
Likewise, thaw reagents from cold storage at room temperature. Set heat block
at 65 °C and set the hybridization oven to 45 °C.
2. Place the 20× hybridization controls tube into the 65 °C heat block and incubate
5 min.
3. Mix the hybridization cocktail for each GeneChip. Use the following reaction
mix: 3-7 μg (˜50 μL) of cDNA, 3.3 μL of control oligonucleotide B2 (3 n M ),
10 μL of 20× hybridization controls, 2 μL of herring sperm (10 mg/mL), 2 μL of
BSA (50 mg/mL), 100 μL of 2× hybridization buffer, and 32.7 μL of H 2 O for a
final volume of 200 μL.
4. Place the GeneChips face down on the bench. Place a 200-μL pipette tip in one
of the septa. Using the other septum, add 200 μL of the appropriate hybridization
cocktail.
5. Place the GeneChips into the hybridization oven (45 °C) for 16 h at 60 rpm.
3.9. Processing Affymetrix GeneChips
Begin processing the Affymetrix GeneChips immediately following
hybridization.
1. Turn on the fluidics station, while also assuring the tubing is placed into wash
bottles A and B, water, and waste.
2. Turn on the workstation and open GCOS.
3. Click on the “fluidics” icon and prime the workstation(s) intended for use.
4. For each chip, using an amber tube, mix 600 μL of 2× stain buffer, 48 μL of
BSA (50 mg/mL), 12 μL of streptavidin phycoerythrin (SAPE 1 mg/mL), and
540 μL of distilled H 2 O. This is the SAPE solution mix.
5. Remove 600 μL of the SAPE solution and place into another amber tube.