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