Biology Reference
In-Depth Information
9. Hybridization oven 640 (Affymetrix).
10. Surfact-Amps 20 (Tween-20), 10% (Pierce Chemical, Rockford, IL).
11. 12× 2-(N-morpholino) ethanesulfonic acid (MES) stock buffer (1.22
M
MES,
0.89
M
Na
+
): 64.61 g of MES hydrate, 193.3 g of MES sodium salt, and 800
mL of nuclease-free water. Adjust the pH to 6.5-7.0, mix and adjust volume
to 1000 mL. Filter-sterilize (0.2 μm) and store at 2-8 °C protected from light.
Discard if the solution turns yellow.
12. 2× hybridization buffer (50 mL): 8.3 mL of 12× MES stock buffer, 17.7 mL of
5
M
NaCl, 4.0 mL of 0.5
M
EDTA, 0.1 mL of 10%-Tween 20, and 19.9 mL
nuclease-free water. Store hybridization buffer at 2-8 °C protected from light.
2.11. Washing and Staining of S. aureus Affymetrix GeneChips
1. Nuclease-free water (Ambion).
2. BSA solution, 50 mg/mL (Invitrogen Life Technologies).
3. NaCl, 5
M
, RNase/DNase free (Ambion).
4. R-Phycoerythrin Streptavidin (Molecular Probes/Invitrogen Life Technologies).
5. Fluidics Station 450 (Affymetrix).
6. 20× Saline-Sodium Phosphate EDTA buffer (SSPE) (BioWhittaker Molecular
Applications, Walkersville, MD/Cambrex): 3
M
NaCl, 0.2
M
NaH
2
PO
4
, 0.02
M
EDTA).
7. Goat IgG, Reagent Grade (Sigma-Aldrich, St. Louis, MO).
8. For 10 mg/mL stock reagent, resuspend 50 mg of goat IgG in 5 mL 150 mM
NaCl. Store at 4 °C.
9. Biotinylated anti-streptavidin goat antibody (Vector Laboratories, Burlingame,
CA).
10. Surfact-Amps 20 (Tween-20), 10% (Pierce Chemical, Rockford, IL).
11. Wash Buffer A, non-stringent wash buffer (6× SSPE, 0.01% Tween-20): 300
mL of 20× SSPE, 1.0 mL of 10% Tween-20, and 699 mL of water. Filter buffer
using a 0.2-μm filter and store at room temperature.
12. Wash Buffer B, stringent wash buffer (100 m
M
MES, 0.1
M
Na
+
, 0.01% Tween-
20): 83.3 mL 12× MES stock buffer, 5.2 mL 5
M
NaCl, 1.0 mL 10% Tween-20,
and 910.5 mL water. Filter buffer using a 0.2-μm filter and store at 2-8 °C
protected from light.
13. 2× Stain Buffer (100 m
M
MES, 1
M
Na+, 0.05% Tween-20): 41.7 mL 12× MES
stock buffer, 92.5 mL 5
M
NaCl, 2.5 mL 10% Tween-20, and 113.3 mL water.
Filter buffer using a 0.2-μm filter and store at 2-8 °C protected from light.
2.12. GeneChip Scanning and Conversion of Image Files
1. Affymetrix GeneChip Scanner 3000 7G Autoloader capable of high-resolution
scanning (Affymetrix).
2. Affymetrix GeneChip Operating Software (GCOS) and the GeneChip Scanner
3000 high-resolution scanning patch (Affymetrix).
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