Paul.xu: Created page with "1. Harvest adherent cells into serum-free medium by gentle scraping. Most laboratory-scale preps (<10^9 cells) can be harvested into a total pooled volume of 200 ml or less. C..."

2012-04-15T17:56:56Z

Created page with "1. Harvest adherent cells into serum-free medium by gentle scraping. Most laboratory-scale preps (<10^9 cells) can be harvested into a total pooled volume of 200 ml or less. C..."

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1. Harvest adherent cells into serum-free medium by gentle scraping. Most laboratory-scale preps (<10^9 cells) can be harvested into a total pooled volume of 200 ml or less. Cells grown in suspension are harvested directly by low-speed centrifugation.

2. Pellet the AAV-containing cells in a 250-ml centrifuge tube at 200 × g for 10 min and remove the supernatant

3. Rinse the cell pellet by suspension in 10 ml of ice-cold PBS and centrifuge as step 2.

4. Resuspend the washed cell pellet in 8 ml of serum-free culture medium and transfer to a 50-ml polypropylene conical tube.

5. Lyse cells by freeze-thawing three times using dry ice and 37 °C water bath.

6. Spin down cellular debris by centrifugation at 3000 × g for 10 min.

7. Using a pipette, carefully transfer the AAV-containing supernatant to a fresh 50-ml conical tube. Take care not to disturb the pelleted cellular debris as transfer of debris may hamper subsequent filtration.

8. To digest non-encapsidated DNA, add Benzonase® nuclease to a final concentration of 150 units nuclease per milliliter of lysate and incubate the lysate in a 37 °C water bath for 1 h.

9. Filter the nuclease-treated lysate through a 0.2-μm syringe-driven filter unit.

10. Add 1/12th volume of the 50% PEG 8000 stock solution to the Oak Ridge tube (for a final concentration of approximately 4% PEG).

11. Place the tube on ice for at least 1 h.

12. Collect the PEG precipitate by centrifugation for 30 min at 12,000 × g.

13. Discard the supernatant.

14. Spin the tube briefly at 120 × g and remove residual PEG solution.

15. Resuspend the precipitate in 8 ml of buffer A by pipetting. The suspended precipitate (which will remain opaque) should be mixed until particulates are no longer visible.

16. Attach an anion exchange column SOURCE 15Q ion-exchange column (Tricorn 10/100 column, ̃8 ml bed volume) to FPLC.

17. Wash column with 3 CV of low-salt buffer (buffer A) followed by 2–3 CV of high-salt buffer (buffer B), and then a final equilibration with at least 3 CV of low-salt buffer at a flow rate of 4 ml/min.

18. Filter the resuspended PEG precipitate using a large diameter (33 mm) 0.2-μm syringe filter.

19. Load into FPLC using 10 ml “Superloop”.

20. Wash the column with buffer A until no detectable absorbance at 280 nm.

21. Elute with a linear salt gradient (0 to 50% buffer B over a course of 20 ml (10 min at 2 ml/min).

22. Beginning 3 ml after the start of the salt gradient, collect the eluate as 1-ml fractions over the course of the gradient.

23. To identify the AAV-positive peak fractions, analyze gradient fraction samples by qPCR

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[[Category:Protocols]]

AAV Purification by Chromatography - Revision history

Paul.xu: Created page with "1. Harvest adherent cells into serum-free medium by gentle scraping. Most laboratory-scale preps (<10^9 cells) can be harvested into a total pooled volume of 200 ml or less. C..."