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Adenovirus Vector Construction Manual Document: (03/00A) 01-1 341 Bering Avenue, Toronto, Canada M8Z 3A8 Tel: 416-234-1624 Fax: 416-234-1626 customer.service@ Ad Vector Construction Manual ________________________________________________________________ TABLE OF CONTENTS _______________________________________________________ PRODUCT INFORMATION 3 GROWTH OF 293 CELLS 4 GENERAL COMMENTS4 PROCEDURE FOR SPLITTING CELLS 4 SOLUTIONS 4 GROWTH MEDIUM 5 FREEZING CELLS FOR STORAGE IN LIQUID N2. 5 THAWING FROZEN CELLS. 5 CSCL-BANDED PLASMID DNA PREPARATION FOR COTRANSFECTIONS 6 PROTOCOL 6 BUFFERS 7 COTRANSFECTION OF 293 CELLS WITH PLASMID DNA FOR VECTOR RESCUE BY THE ADMAXTM METHOD 9 PROTOCOL 9 NOTES 9 SCREENING ADENO PLAQUE ISOLATES FROM VECTOR RESCUES 10 PROTOCOL 10 PREPARATION OF HIGH-TITER VIRAL STOCKS (CRUDE LYSATES) FROM CELLS IN MONOLAYER 12 PROTOCOL 12 SOLUTIONS 12 PREPARATION OF HIGH-TITER CRUDE VIRAL STOCKS FROM CELLS IN SUSPENSION 13 PROTOCOL 13 SOLUTIONS 13 PREPARATION OF PURIFIED HIGH-TITER VIRAL STOCKS BY CSCL BANDING 14 PROTOCOL 14 MATERIALS 15 SOLUTIONS 15 REFERENCES 16 RANSFECTION 1. DNA T 16 2. 293 CELLS16 3. INFECTIVITY OF CIRCULAR FORMS OF AD DNA16 4. THE TWO PLASMID METHOD FOR VECTOR RESCUE 17 5. PACKAGING CAPACITY OF AD VECTORS 17 6. AD VECTORS AS VACCINES 17 7. METHODS ARTICLES. 18 8. GENERAL 18 ADMAXTM PLASMID MAPS AND TECHNICAL ASSISTANCE 19 ________________________________________________________________ (03/00A) 01-1 2 of 19 Ad Vector Construction Manual ________________________________________________________________ Product Inf