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
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