Transfection of Proviral Constructs

1. 6- and/or 10-cm tissue-culture dishes.

2. Sterile 4-mL polypropylene round bottom tubes.

5. 2X HEPES buffered saline (HBS): 280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, 12 mM dextrose, 50 mM HEPES. Adjust pH to 7.05 with 0.5 N NaOH and filter-sterilize. Aliquot and store at -20°C.

6. 2 M CaCl2: filter-sterilize, and store in aliquots at -20°C.

8. 15% (v/v) glycerol in HBS: filter-sterilized.

9. Stock solution of 2 mg/mL polybrene (Sigma, St. Louis, MO) in H2O: filter-sterilize, and store in aliquots at -20°C. Polybrene is harmful by inhalation. Therefore, adequate safety precautions should be taken.

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