Igem2010/Main/Homology Based/October

From 2010.igem.org

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==01/10/2010==
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* 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

Revision as of 12:10, 26 October 2010

Contents

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


August
MTWTFSS
1
2345678
9101112131415
16171819202122
23242526272829
3031
September
MTWTFSS
12345
6789101112
13141516171819
20212223242526
27282930
-
October
MTWTFSS
010203
04050607080910
11121314151617
18192021222324
25262728293031
-