Editing AAV-Production-293T (section)

= Purification of Recombinant Rep Protein  =

<br>1. Inoculate overnight culture of LB with BL21 cells. Grow at 37°C in the presence of 25 ÎŒg/mL kanamycin and 100 ÎŒg/mL ampicillin. 

2. Use overnight culture to inoculate 1 L of LB media and grow cells to an OD600 of 0.8. 

3. Induce cells with 0.1 mM IPTG for 1 h at 37°C. 

4. Pellet cells at 6000g for 20 min at 4°C, wash in 50 mM NaPO4 monobasic pH 8.1, 1 M NaCl and repellet. 

5. Resuspend in 20 mL of column buffer. Freeze/thaw twice in dry-ice/ethanol bath. 

6. Add lysozyme to 1 mg/mL and incubate on ice for 30 min. 

7. Sonicate for 30 s on ice (output 6 on a Branson Sonifer 250, VWR Scientific). 

8. Spin at 14,000g at 4°C for 30 min and collect supernatant. 

9. Apply 5 mL of extract to a 2-mL Ni-NTA agarose column (QIAGEN) equilibrated in column buffer with 20% glycerol at a flow rate of 0.5 mL/min. 

10. Wash with 5 vol column buffer supplemented with 100 mM imidazole and 20% glycerol. 

11. Elute in an ascending linear gradient of 0.1 to 1.0 M imidazole and identify fractions containing Rep68H6 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. 

12. Pool peak fractions and dialyze into 25 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM DTT, 0.1% Tween-20, 20% glycerol and store at â80°C.