Analysis of Staphylococcus aureus Gene Expression During PMN Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 106

dATP, dCTP, dGTP, and dTTP to 600 μL nuclease-free water. Store this solution
at -20 °C in a non-frost-free freezer.
2. Random primers, 3 μg/μL (Invitrogen, Life Technologies). For 75 ng/μL of
working stock, mix 25 μL of 3 μg/μL random primers with 975 μL of nuclease-free
water. Store this solution at -20 °C in a non-frost-free freezer.
3. Superscript II or Superscript III Reverse Transcriptase (Invitrogen Life
Technologies).
4. 5× 1st Strand Buffer (Invitrogen Life Technologies).
5. 100 m M DTT (Invitrogen Life Technologies).
6. SUPERase·In, (Ambion).
7. Nuclease-free water (Ambion).
2.6. Removal of RNA
1. 1 N NaOH solution (VWR Scientific Products, West Chester, PA).
2. 1 N HCl solution (VWR Scientific Products).
2.7. Purification of cDNA
1. QIAquick PCR Purification Kit (Qiagen).
2.8. cDNA Fragmentation
1. 10× One-Phor-All Buffer (GE Healthcare Biosciences, Piscataway, NJ).
2. DNase I (GE Healthcare Biosciences, Piscataway, NJ).
2.9. Terminal Labeling of Fragmented cDNA
1. Enzo BioArray Terminal Labeling Kit (Enzo Biochem, Inc. New York, NY):
5× reaction buffer, 10× CoCl 2 , biotin-ddUTP, and terminal deoxynucleotide trans-
ferase.
2.10. Hybridization of S. aureus cDNA to Affymetrix GeneChips
1. Nuclease-free water (Ambion).
2. Sterile, RNase-free microcentrifuge vials (1.7-mL, Ambion).
3. Aerosol-resistant micropipette tips, RNase-free, non-stick (Ambion).
4. Bovine serum albumin (BSA) solution, 50 mg/mL (Invitrogen Life
Technologies).
5. Herring sperm DNA (Promega Corporation, Madison, WI).
6. Control Oligo B2, 3 n M , including 20× hybridization controls (Affymetrix).
7. NaCl, 5 M , Rnase/Dnase free (Ambion).
8. Tough-Spots label dots (Diversified Biotech, Boston, MA).