Editing AAV-Production-293T-2 (section)

=== <br> AAV &amp; Adeno  ===

Day 0 

Plate Ad-293 cells at 3e5 per well in 6 well plate (adeno) or 5e5 per 6 cm dish (AAV) 

Day 1 

Replace media 

Adenovirus 

A.&nbsp; 1 ug of PacI digested pAdE construct in 250 ul OPTI-MEM without serum 

B.&nbsp; 3 ul of Lipofectamine 2000 in 250 ul OPTI-MEM without serum, incubate 5 minutes at RT 

Gently mix A and B together and incubate for 20 minutes at RT.Â May be slightly cloudy. 

Add complexes directly to a single well of six well plate. 

48 hours post-transfection,Â trypisinize each well and transfer to a 10 cm plate. 

Replace media every 2-3 days until 80% CPE 

AAV 

A.&nbsp; 1.5 ug of ITR AAV vector construct and 4.5 ug of packaging vector (DF1-6)Â in 500 ul OPTI-MEM without serum.Â (3:1 ITR:packaging vector ratio) 

B.&nbsp; 15 ul of Lipofectamine 2000 in 500 ul OPTI-MEM without serum, incubate 5 minutes at RT 

Gently mix A and B together and incubate for 20 minutes at RT.Â May be slightly cloudy. 

Add complexes directly to a single 6 cm dish. 

48 hours post-transfection, check for fluorescence and harvest cells with cell scraper.Â 80% of virus will be in cell pellet. 

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Size of culture vessel (surface area) 

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Approximate cells/plate for transfection 

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Volume of media/well 

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Volume of trypsin 

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24 well plate (2 cm2) 

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1 x 105 

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500 ul 

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|-
| width="153" valign="top" | 
6 well plate (35mm, 10 cm2) 

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3 x 105 

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2 ml 

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300 ul 

|-
| width="153" valign="top" | 
6 cm dish (20 cm2) 

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5 x 105 

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5 ml 

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0.5 ml 

|-
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10 cm dish (60 cm2) 

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2 x 106 

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12 ml 

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1 ml 

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15 cm dish (135 cm2) 

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1 x 107 

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25 ml 

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2 ml 

|}

[[Category:Protocols]]