EasyPrep TM Adeno-Associated Virus Purification Mini Kit Manual (All Serotype) Catalog#: AAV11-00, AAV11-01, AAV11-02 For research use only (October 2013)
Table of Contents Introduction... 4 Storage and Stability... 4 Before Starting... 4 Kit Contents... 5 Safety Considerations 5 Introduction The AAV purification kit is designed for fast and efficient purification all serotypes of raav from AAV infected cell culture. Viral particles can be purified from cell culture of 1 to 2 T 75 flasks per column. The viruses are first applied to a purification column and then further purified and concentrated through a centrifugal filter. Before Starting Familiar with each step by reading this menu and prepare all materials for the procedure. Follow safety regulations for AAV. AAV Purification Protocol...... 6 Trouble Shooting Guide... 9 Limited Warranty 10 EasyPrep TM AAV Miniprep 3 4 EasyPrep TM AAV Miniprep
Kit Components Stability Catalog# AAV11-00 AAV11-01 AAV11-02 Preps 2 10 20 AAV Column 1 5 10 Press-On Cap 1 10 20 Centrifugal filter 2 5 10 Nuclease 10 µl 55 µl 110 μl 10x Nuclease Buffer 100 µl 500 μl 1000 μl Buffer B 10 ml 35 ml 70 ml Buffer P 10 ml 70 ml 140 ml Buffer S 30 ml 100 ml 200 ml Regeneration Buffer 10 ml 20 ml 40 ml The Guaranteed shelf life is 12 months from the date of purchase. Store AAV column at 4 C Safety considerations The AAV infected cell culture and the purified virus can be biohazardous material and can be infectious to human and animals. All protocols MUST be performed under Bio-Safety working conditions for AAV according to regulations. Materials required but not supplied Adeno-associated Virus Purification Protocol Harvest AAV from cell culture (1-2 T 75 flasks) 1. For adherent transfected cells, use a pasteur pipette to remove the culture medium and harvest cells with 3-5 ml PBS. Pellet the cells at 1000 rpm for 10 minutes. Discard supernatant. Resuspend the cells in 3 ml Buffer B. Make sure there s no cell clumps remaining after resuspension. 2. Add 30 μl 100x Nuclease Reaction Buffer and 5 μl Nuclease. Mix well by pipetting and incubate at 37 C for 30 minutes. Centrifuge at 600 x g for 15 minutes, transfer the supernatant to a clean tube, further clarify the supernatant through a 0.45 μm filter unit. Add 1 volume of Buffer P to 4 volume of virus lysate (For example, add 0.75 ml of Buffer P to 3 ml of virus lysate). Mix well and incubate at 4 C for at least 3 hours to overnight. The virus is stable in Buffer P. 3. Centrifuge the sample at 3,000 rpm for 30 minutes (Prepare for step 4 during centrifugation). After centrifugation, carefully aspirate and discard the supernatant. Spin briefly and remove the residual supernatant. The virus containing pellet should be visible. The pellet may appear hazy. Keep the virus on ice. Purification column preparation Standard TC centrifuge Swing bucket rotor. 0.45 μm filter unit Rack holder for column 4. Inverting the AAV column to resuspend the resin inside the column. Put the column into a 15 ml conical tube and centrifuge at 800 rpm for 2 minutes. Tear off the breakoff tip on the bottom of the column and place the column into the 15 ml tube. Loosen the cap to allow buffer drain out from the column by gravity. Once the liquid stops dripping, add 4 ml Buffer S evenly to the column and let it drain out by gravity. Once all the Buffer S get into the resin, stop the gravity flow by capping the bottom tip of the column with a Press-on cap, to prevent the column from drying. EasyPrep TM AAV Miniprep 5 6 EasyPrep TM AAV Miniprep
5. Dissolve the pellet from step 3 with 4 ml Buffer S by pipetting and vortexing. Split the sample into 2-3 Eppendorf microcentrifuge tubes, and spin at 10,000 rpm for 2 min at 4 C. Transfer the clear supernatant to a clean 15ml conical tube. 6. Load the clear supernatant from step 5 to the reservoir of a centrifugal filter and centrifuge at 3,000 rpm for 10-20 minutes till around 300 μl of sample remains in the reservoir. Transfer the sample to a clean micro-centrifuge tube. Wash the reservoir with 100 μl Buffer S and transfer the sample to the same microcentrifuge tube. Load the sample to the purification column 7. Slowly and evenly add the sample (about 400 μl) collected from step 6 drop by drop to the AAV column, and let it flow into the resin by gravity. Once the sample gets into the resin, discard the flow-through and put back the conical tube under the column, proceed to step 8 immediately. Elute AAV from the purification column Swing Bucket Rotor (3,000 rpm, 4 ml starting volume) 10. The purified virus is ready for downstream applications. Aliquot and store the purified virus at -80 C. Regeneration of the column 35 o Fixed Angle Rotor Spin time Volume left Spin time Volume left 15 min 176 μl 10 min 97 μl 20 min 76 μl 15 min 54 μl 25 min 58 μl 20 min 35 μl Upon completion of the purification, add 3 ml Regeneration Buffer to the column and let the buffer passes through the column by gravity flow. Wash the column by 6 ml PBS, let the PBS pass through the column by gravity flow. Once the liquid stops dripping, fill the column with 2 ml PBS. Press on the cap to the bottom. Screw on the cap and wrap the column with parafilm in a zip block bag and store at 4 C. 8. Add 4 ml Buffer S evenly to the column and collect 4 ml of the flow-through. The virus is in the flow through liquid. Concentration 9. Apply 4 ml of the sample to a reservoir of a centrifugal filter and centrifuge at 3,000 rpm for 10-30 minutes till 200-300 μl remains in the reservoir. Pipet the solution up and down several times in the reservoir and transfer the virus containing solution to a clean vial. Note: A swing bucket rotor is preferred. Fixed angle rotor requires higher speed of 7000 rpm for 15-20 minutes. Note: Time for centrifugation may vary for different type rotors. Always centrifuge less time and check the liquid level, repeat centrifuge to get to the expected volume. EasyPrep TM AAV Miniprep 7 8 EasyPrep TM AAV Miniprep
Trouble shooting Guide Limited use and warranty Problems Slow flow rate caused by air bubbles in the resin bed Slow flow rate caused by invisible bubbles Supernatant very viscous Column clogged after loading sample Solutions Cap the bottom of the column with the press on cap and spin the column at 300 x g for 5 minutes. With the bottom cap on, add degassed water to the resin with a height of 1-2 cm of the solution. Place the entire bottom-capped column in a 15 ml conical tube and centrifuge at 10 minutes at 1,000 x g. Forgot to filter the supernatant through a 0.45 µm filter unit. Resuspend and dissolve the virus pellet completely with Buffer S. Spin down briefly to remove any insoluble debris. This product is intended for in vitro research use only. Not for use in human. This product is warranted to perform as described in its labeling and in Bioland s literature when used in accordance with instructions. No other warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Bioland. Bioland s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of Bioland, to replace the products, Bioland shall have no liability for any direct, indirect, consequential, or incidental damage arising out of the use, the results of use, or the inability to use it product. For technical support or learn more product information, please contact us at (877) 603-8882 or visit our website at www.bioland-sci.com EasyPrep TM AAV Miniprep 9 10 EasyPrep TM AAV Miniprep
Bioland Scientific LLC 14925 Paramount Blvd., Suite C Paramount, CA 90723 USA Tel: (877) 603-8882 Fax: (562) 733-6008 Email: service@bioland-sci.com order@bioland-sci.com Visit our web at www.bioland-sci.com and learn more about Bioland products EasyPrep TM AAV Miniprep