Paul.xu at 16:44, 19 November 2013

2013-11-19T16:44:37Z

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== HiTrap Q HP ==

1. Wash strong anion exchange column HiTrap Q HP (5 ml, Amersham Biosciences) with 25 ml of PBS, pH adjusted to 7.5, flow rate of 5 mL/min. 2. Wash column with 25 ml of PBS-1M NaCl, 5 ml/min. 3. Wash column with 25 ml of PBS, 5 ml/min 4. Mix 0.5 ml concentrated lentivirus with 1 ml starting buffer, spin for 5 min to remove large debris at 6000× g for 5 min. 5. Load sample to column. 6. Wash with 25 ml washing buffer 7. Elute with salt gradient step (0–1 M NaCl) for 10 min, 2.5 mL/min of flow rate, collect 1 ml/tube. 8. Virus peak: about 40 ml 9. Use Centricon to desalt. 

 

1. Transfect 2 x 106 293T cells with each 8 μg V, G, and pLVX construct 2. After 48 h, the supernatant was harvested and filtered through a 0.45-μm pore size filter. 3. Concentrate to 1/100 volume by ultracentrifugation with 50 000 × g at 4°C for 2.5 h using a SW28 rotor. 4. Resuspend virus particle pellet in PBS and frozen at -80°C until use 5. Wash column with 5 column volumes (25 mL) of starting buffer (PBS adjusted pH at 7.5) at a flow rate of 25 mL/min. 6. Wash column with the elution buffer (PBS, pH 7.5 containing 1 M NaCl) at a flow rate of 25 mL/min. 7. Equilibrate column with the starting buffer at 2.5 mL/min for 10 min. 8. Mix concentrated virus vector (0.5 mL) suspended with 2.5 ml of starting buffer 9. Spin at 6000 × g for 5 min to remove large particles. 10. Load virus suspension to column. 11. Wash with starting buffer. 12. Elute with NaCl gradient (0–1 M NaCl) for 10 min, at 2.5 mL/min. 13. Concentrate the virus using Centricon Plus-20, cut-off 100 000 molecular weight; Millipore, Bedford, MA, USA). The membrane should be pre-sterilized by washing with 70% ethanol 

[[Category:Lentivirus]] [[Category:Protocols]]

Lentivirus Purification Anion Exchange Column - Revision history

Paul.xu at 16:44, 19 November 2013