Virus Harvest AAV 15 cm 2

Transcription

1 Virus Harvest AAV 15 cm 2 1) Gently scrape cells from plate in 10 ml of media, place remaining 10 ml of media in plate lid. 2) Once cells are removed from plate, wash plate by pipetting 20 ml of media over whole plate and save cell/supernatant in 250 ml conical. 3) Wash all plates with 1x PBS and place remaining cell/pbs supernatant in existing 250 ml conical 4) Spin cells down for 15 minutes at 2000 rpm then aspirate off media 5) Resuspend cells in lysis buffer (10 ml for every ten 15 cm 2 plates). 6) Place in -80 C freezer for at least one hour. Virus Harvest 6-cm 2 dish for PAGE 1) Aspirate Media 2) Resuspend cells in 1 ml of 1x PBS on bench 3) Spin down cells at low speed (5 Kprm) 4) Aspirate 1 ml of 1x PSB 5) Resuspend cells in µl of Lysis buffer with protease inhibitors 6) Put on ice for 30 minutes 7) Spin down at high speed 8) Retain Lysis buffer containing proteins in supernatant 9) Place supernant in new tube and discard pellet from previous tube 10) Freeze until further use 11) When ready to use, add 2x loading dye to appropriate amount and heat at 95 for ten minutes. Virus Purification 1) Thaw transfected virus from -80 C in 37 C water bath 2) Spin at 2 K for 15 minutes 3) Pipette off supernatant (virus in supernatant) and place in new tubes. Discard debris in bottom of tube. 4) Add 1 µl of Benzonase (located in -20 C freezer, door) 5) Incubate for minutes in 37 C water bath 6) Repeat step #2 and #3 to ensure all debris has been discarded

2 Setting up the Pump 1) Place 250 ml conical tubes on either side of pumps in tube holders. 2) Place capillary tubes in holders on pump 3) Place capillary tube sticks in capillary tubes a. Red sticks go from source tube to pump b. Blue Sticks go from pump to destination tube 4) Pour sterile water in source 250 ml conical tube, and then flush system. * Tubes must be completely filled with water initially to ensure that the operation is functional. Transferring Iodixanol Iodixanol: 15% (5.5 ml/tube), 25% (5.5 ml/tube), 40% (4 ml/tube) and 60% (5 ml/tube); Shake to agitate prior to pipetting. 1) Add virus from Sarstedt 15 ml tubes to respective Large 70 NVT Beckman tubes with 2 ml pipette SLOWLY, bubbles can not be formed. 2) Ensure that all Red tubes are LEVEL from source tube 3) Place capillary tubes in respective Large 70 NVT Beckman tubes 4) Press start button and fill with appropriate amount of 15% Iodixanol (i.e. 22 ml for 4 tubes 5.5 ml * 4 tubes = 22 ml, but add 23 ml to be sure) until ½ ml is left in bottom of source (22.5 ml is transferred through) *MAKE SURE TO STOP at ½ ml 5) Add 22 ml of 25% Iodixanol (4 tubes * 5.5 ml) to source tube, press start, and stop again at ½ ml. *MAKE SURE TO STOP at ½ ml 6) Add 22 ml of 40% Iodixonal (4 tubes * 5.5 ml) to source tube, press start, and stop again at ½ ml. *MAKE SURE TO STOP at ½ ml 7) Add 16 ml of 60% (stock) (4 tubes * 4 ml) to tube, press start, and stop again at ½ ml. 8) If less than 10 ml, we must add lysis buffer to bring levels equal and to top of Beckman tube. Do this with 5 ml syringe and do NOT form bubbles. 9) Flush system immediately with sterile water to ensure cleanliness

3 1) Spinning at 69 K for 1 hour at 18 C in NVT 70 TI Rotor 2) Carefully transport Rotor to hood for opening 3) Place Beckman tube in holder * Extracting Virus from 4 gradients is as follows. Gradients are Lysis Buffer (Top), 15 % (2 nd ), 25 % (3 rd ), Empty Capsids (4 th ), 40 % (3 rd ), 60 % Bottom. Extract section above 60 % to 40 %; this is a continuous gradient therefore there will be more AAV closer to the 60%. Below the 25 % but above the 40 %/AAV are the empty capsids. 4) Extract empty capsids with one syringe and 40 % gradient (virus) with another a. If same serotype is used for all virus preparations use same syringe b. Each virus extraction will be approximately 4-5 ml; do not extract empty capsids in an effort to maximize virus extraction 5) Remove needle and discharge virus to appropriately labeled 15 ml Sarstedt tubes 6) Store at 4 C Virus Purification with Column Pump Set-Up 1) Attach the appropriate adapters to clear leads 2) Clamp 60 ml syringe to clamp above pump 3) Attach syringe adapter to syringe and column adapter to column 4) Plug pump in but keep switch to off position

4 Function of Buffers all ph 8.5 Buffer A Basically water, 15 mm NaCl, 20 mm TRIS Buffer B Used to elute any proteins or impurities, 1 M NaCl, 20mM TRIS Buffer C Removes protein; allows virus eluted exclusively; 20 mm Tris, 355 mm NaCl Virus Elution 1) 30 ml Buffer A, Flow Rate 10, General cleaning and equilibrating 2) 25 ml Buffer B, Flow Rate 10, Flushes impurities 3) 50 ml Buffer A, Flow Rate 10, Washes solution 4) Dilute prep 3x in 50 ml Sarstedt (i.e. 8 ml Ric-3; 16 ml Buffer A = 24 ml Dilution), flow rate 5. *Virus binds to column in this step 5) Buffer A ml, Flow Rate 5, washes previous step 6) Place protein concentrating tube under column in holder to catch elution 7) Buffer C 5 ml, Flow Rate 5, elutes contents of column to concentrating column. *Theoretically, the first 1-2 ml elutes all of the virus, the rest confirms elution 8) Clean system with bleach and water at Flow Rate of 10 Virus Concentration * It is likely that the elution will be all the way to the top of the 15 ml concentrator tube and will have to be spun down for 3 minutes at 2K RPM in order for Sodium Citrate to be added. 1) Fill Sodium Citrate to 15 ml mark in 15 ml tube and spin down for 3 minutes at 2K RPM. 2) Repeat this procedure three times. 3) Spin at approximately 1 K for one minute intervals. a. Carefully watch the level within the column b. Spin until desired volume (i.e. 250 µl) is reached 4) Transfer desired volume to Eppendorf tube and store at 4 C

5 Put 8 µl aliquot for titer in Eppendorf tube Put rest of purified virus for stock Heparin Purification 1) Attach column to stand and place stoppers in bottom 2) Mix Heparin non-vigorously 3) Place beaker under column 4) Add 3 ml of Heparin/Buffer a. Volume will be 1.5 ml when buffer has run out 5) Add 3 ml of 1xTD/1xNaCl a. Wait minutes so that Heparin impurities are cleaned 6) Wash NaCl out with 1xTD a. This ensures that all NaCl has washed out