Editing AAV-Production-293T (section)

= Protocol 1  =

1. Culture 10<sup>7</sup> 293T cells in ten 15cm culture dishes one day prior to transfection. 

2. Add 300 ÎŒg of AAV vector plasmid and 1400ÎŒg of packaging plasmid to 10 mL of DMEM. 

3. Add GeneExpresso Max to 10 ml DMEM. 

4. Add GeneExpresso Max in DMEM to DNA in DMEM, mix and incubate for 20 minutes at room temperature. 

5. Add 2 ml transfection mixture to each plate 

6. Incubate cells for 37ºC and 5% CO2. 

7. Remove transfection media after 15 hours and add fresh culture media. 

8. Harvest cells two days after transfection. 

9. Pellet cells by centrifugation and resuspend in 15mL of 50mM Tris (pH 8.5) containing 150mM NaCl (or PBS, pH 7.5, 1 mM, MgCl2 and 10 mg/ml DNase I) 

10. Lyse cells by three freeze-thaw cycles (dry ice/100% ethanol bath and 37°C water bath). 

11. Add benzonase to lysate at a final concentration of 50U/mL and incubated at 37ºC for one hour. 

12. Prepare iodixanol gradient in 35mL ultracentrifuge tubes, overlaying (from bottom to top) 5mL of 60% iodixanol, 5mL of 40% iodixanol, 6mL of 25% iodixanol and 9mL of 15% iodixanol. 

13. Add cell lysate gradient tube and&amp;nbsp; seal the tube sealed with rotor cap accessory. 

14. Spin at 350000g for two hours. 

15. Remov AAV from the clear 40% layer by making an air hole on the top of the tube and removing the layer with a needle. 

16. Purify AAV using HiTrap heparin&nbsp;column (or heparin-agarose beads column, Sigma, H-6508)&nbsp; by FPLC. 

17. Make 200 µl aliquots and store in a â80ºC freezer.<br> 

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