Igem2010/Main/Homology Based/October

01/10/2010

• 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.

07/10/2010