Harvesting of the 50 plates (15 cm) from the previous day and picking of 50 clones were done, and the collected bacteria were incubated in a 700 mL LB culture for 3 hours with shaking, after which a megaprep (Invitrogen) was done.
mini-preps of the 50 clones were done, and 10 samples were sent for sequencing.
02/10/2010
Transfection of 13 T flasks (150 cm2, Hek 293 cells) was done, with the plasmid DNA purified the previous day (the shuffled cap genes library) and an adeno-helper plasmid, 35 micrograms from each plasmid for each flask, using HBSS for the transfection. A GFP control for transfection was also used.
Analysis of the sequencing results for the 10 samples identified good shuffling of the cap genes. More mini-preps were inoculated for the same 50 clones.
03/10/2010
Mini-preps for the samples from the previous day were done.
05/10/2010
Harvesting of the cells for virus production was done by:
- Washing the flasks with the medium they contain, and collecting the media from 13 flasks in 500 ml corning conical centrifuge tubes.
- The cells were pelleted by centrifuging at 1500 rpm for 15 min at 4 ͦC
- Discard supernatant
- Washing: Resuspend the cells in 1X PBS and transfer to a 50 ml falcon
- Pellet the cells again by centrifuging at 1500 rpm for 15 min at 4 ͦC
- Discard supernatant
Freeze-thaw cylces for cell rupturing:
- Add 20 ml lysis buffer to the cell pellet
- Put in liquid nitrogen for 5 min
- Thaw at 37ͦC
The cycles were repeated for 5 times, and the sample was frozen for next day.
06/10/2010
The preparation for virus purification using iodixanol gradient was initiated according to the following:
- The sample from previous day was sonicated in a sonication bath for 1 min 20 sec.
- 50 units/ml benzonase were added to destroy RNA and DNA from non-AAV sources, which could later interfere with qPCR.
- The sample was incubated at 37 ͦC for 30 min, and vortexed every 10 min.
- The sample was then centrifuged for 15 min at 3270 xg
- The supernatant was transferred into a new 50 ml falcon
Virus purification using iodixanol gradient centrifugation:
- A Pasteur pipette was plugged into a Beckman quick-seal centrifuge tube
- Using a 1000 µl pipette, 20 ml of the virus suspension were added the gradient was poured through the Pasteur pipette in the following order:
- 7 ml 15% Iodixanol solution
- 5 ml 25% Iodixanol solution
- 4 ml 40% Iodixanol solution
- 4 ml 60% Iodixanol solution
- The Pasteur pipette was carefully removed and the tube was sealed. A tare tube was prepared in the same way.
- Ultracentrifugation of the sample was carried out at 50,000 rpm for 2:30 hours at 4ͦC.
- The virus-containing phase is the 40% iodixanol phase, which was sucked out using a syringe and a needle. The AAV sample was divided into aliquotes, one of them stored at 4 ͦC for qPCR the next day, and the rest were frozen in liquid nitrogen then transferred to -20 ͦC.
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