Paul.xu at 17:21, 10 August 2012

2012-08-10T17:21:20Z

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

 1. Transform E. coli BL21 (DE3) pGro7 cells (Stratagene) with Zinc-finger nuclease plasmid. 2. Grow a single colony in 10 ml LB medium with kanamycin, chloramphenicol and 1% glucose. 3. Grow bacteria overnight at 37 °C with shaking. 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. 6. Add 0.1 mM IPTG and culture for 4 hs. 7. Havest cells by centrifugation. 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). 9. Lyse cells by sonication. 10. Spin and collect supernatant. 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). 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. 2. Incubate cells at 37 °C with shaking until the OD600 reached 0.4. 3. Incubate at room temperature with shaking until the OD600 reaches 0.9. 4. Add 0.4 mM IPTG and 4 mM arabinose and incubate for 16 h. 5. Spin down cells by centrifugation. 6. Resuspend the pellet in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, pH 8.0). 7. Lyse cells by sonication. 8. Spin at 10,000 rpm for 20 min at 4 °C. 9. Wash inclusion bodies with lysis buffer. 10. Spin at 10,000 rpm for 20 min at 4 °C. 11. Repeat step 9 to 10 twice. 12. Wash pellet with cold water then PBS and each time spin down pellet. 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. 14. Spin for 10 min at 10000 rpm. 15. Collect supernatant and purify protein using Ni beads. 

 

== Transduction ==

Dilute ZFNs purified from the soluble fraction with serum-free medium containing 90 μM ZnCl2 at pH 7.4. Dilute ZFNs purified from inclusion bodies with serum-free medium containing 100 mM L-arginine and 90 μM ZnCl2 at pH 7.4. 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. 

[[Category:Protocols]]

Zinc Finger Nuclease Purification - Revision history

Paul.xu at 17:21, 10 August 2012