Editing Zinc Finger Nuclease Purification

== '''Purify from soluble fraction''' ==

<br>1. Transform E. coli BL21 (DE3) pGro7 cells (Stratagene) with Zinc-finger nuclease plasmid. <br>2. Grow a single colony in 10 ml LB medium with kanamycin, chloramphenicol and 1% glucose.<br>3. Grow bacteria overnight at 37 °C with shaking. <br>4. The following day, add 10 ml overnight culture bacteria to 1 L of LB medium supplemented with 100 μM ZnCl2, 50 μg ml−1 kanamycin, 100 μg ml−1 chloramphenicol and 0.2% glucose was inoculated with 5 ml of the overnight culture and incubate at 30 °C with shaking until OD600 is 0.4. 

5. Incubate at room temperature with shaking until the OD600 reaches 0.8.<br>6. Add 0.1 mM IPTG and culture for 4 hs.<br>7. Havest cells by centrifugation.<br>8. Resuspend cells in Buffer A (lysis buffer, 50 mM Tris-HCl, 500 mM NaCl, 100 μM ZnCl2, 1 mM PMSF, 1 mM MgCl2, 1 mM β-mercaptoethanol, 10 mM imidazole, 0.2% Triton X-100 and 10% glycerol, pH 8.0). <br>9. Lyse cells by sonication.<br>10. Spin and collect supernatant.<br>11. Purify ZFN proteins using Ni-NTA agarose resin and elute with Buffer B (500 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, 100 μM ZnCl2 and 10% glycerol at pH 8.0).<br>12. Concentrate protein by dialyzing against storage buffer (20 mM HEPES, 90 μM ZnCl2, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, pH 7.4). 

== '''Purify from Inclusion body'''  ==

1. Inoculate 500 ml of LB medium with 5 ml of the overnight culture.<br>2. Incubate cells at 37 °C with shaking until the OD600 reached 0.4.<br>3. Incubate at room temperature with shaking until the OD600 reaches 0.9. <br>4. Add 0.4 mM IPTG and 4 mM arabinose and incubate for 16 h.<br>5. Spin down cells by centrifugation.<br>6. Resuspend the pellet in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, pH 8.0). <br>7. Lyse cells by sonication.<br>8. Spin at 10,000 rpm for 20 min at 4 °C.<br>9. Wash inclusion bodies with lysis buffer.<br>10. Spin at 10,000 rpm for 20 min at 4 °C.<br>11. Repeat step 9 to 10 twice.<br>12. Wash pellet with cold water then PBS and each time spin down pellet.<br>13. Incubate pellet with solubilization buffer (PBS, 500 mM l-arginine, 90 μM ZnCl2, 1 mM MgCl2, pH 7.4) for 3 h at 4 °C.<br>14. Spin for 10 min at 10000 rpm.<br>15. Collect supernatant and purify protein using Ni beads.<br> 

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== Transduction ==

Dilute ZFNs purified from the soluble fraction with serum-free medium containing 90 μM ZnCl2 at pH 7.4. <br>Dilute ZFNs purified from inclusion bodies with serum-free medium containing 100 mM L-arginine and 90 μM ZnCl2 at pH 7.4. <br>At 24 h after seeding, wash cells with serum-free medium and treat with ZFN proteins for 1 h at 37 °C. After treatment, wash cells and incubate 37 °C with serum-containing medium for 24 h before the next treatment, or up to 3 d. <br>

[[Category:Protocols]]