By Elizabeth Ness MacBean Ross, James Ness MacBean, Ross
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Nichols, 1998 103. Pichia Protocols, edited by David R. Higgins and James M. Cregg, 1998 102. Bioluminescence Methods and Protocols, edited by Robert A. LaRossa, 1998 101. Myobacteria Protocols, edited by Tanya Parish and Neil G. Stoker, 1998 100. Nitric Oxide Protocols, edited by Michael A. Titheradge, 1998 99. Human Cytokines and Cytokine Receptors. edited by Reno Debets, 1998 98. Forensic DNA Profiling Protocols, edited by Patrick J. Lincoln and James M. Thomson, 1998 97. Molecular Embryology: Methods and Protocols, edited by Paul T.
Add 3 mL media (DMEM with 10% fetal calf serum) and incubate at 37°C for 4 h (Note 6). 7. Glycerol shock of cells: Aspirate media from the dish and rinse twice with PBS. 5 min at 37°C. Aspirate glycerol-HBS and add 5 mL media and incubate overnight at 37°C. 8. Let the packaging cells grow to confluence (2448 h). Change the media if necessary. 9. Split the cells with trypsin-EDTA and dilute 5:1 to 10:1 and replate in media containing G418 (5001000 mg/mL) or the appropriate selection antibiotic (Note 7).
Many different cell lines are available (see http://www/atcc/org for details). The packaging cell line described in these experiments is PA317 (ATCC CRL-9078). 4 Materials Needed for Vector Construction and DNA Transformation (Note 2) 1. 2 M CaCl2. 2. 5 mM Na2HPO4 . 2H2O, 12 mM dextrose, 50 mM HEPES) stored in aliquots at -20°C. 3. Sterile glycerol. 4. Sterile vacuum grease. 5. Sterile glass cloning rings. 6. Appropriate restriction enzymes, modifying enzymes, and buffers. 7. Agarose, electrophoresis equipment, and photographic supplies.