TUDelft/15 October 2008

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July
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August
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September
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[http://2008.igem.org/TUDelft/1_September_2008 1] [http://2008.igem.org/TUDelft/2_September_2008 2] [http://2008.igem.org/TUDelft/3_September_2008 3] [http://2008.igem.org/TUDelft/4_September_2008 4] [http://2008.igem.org/TUDelft/5_September_2008 5] [http://2008.igem.org/wiki/index.php?title=TUDelft/6_September_2008&action=edit 6] [http://2008.igem.org/wiki/index.php?title=TUDelft/7_September_2008&action=edit 7]
[http://2008.igem.org/TUDelft/8_September_2008 8] [http://2008.igem.org/TUDelft/9_September_2008 9] [http://2008.igem.org/TUDelft/10_September_2008 10] [http://2008.igem.org/TUDelft/11_September_2008 11] [http://2008.igem.org/TUDelft/12_September_2008 12] [http://2008.igem.org/wiki/index.php?title=TUDelft/13_September_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=TUDelft/14_September_2008&action=edit 14]
[http://2008.igem.org/TUDelft/15_September_2008 15] [http://2008.igem.org/TUDelft/16_September_2008 16] [http://2008.igem.org/TUDelft/17_September_2008 17] [http://2008.igem.org/TUDelft/18_September_2008 18] [http://2008.igem.org/TUDelft/19_September_2008 19] [http://2008.igem.org/wiki/index.php?title=TUDelft/20_September_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=TUDelft/21_September_2008&action=edit 21]
[http://2008.igem.org/TUDelft/22_September_2008 22] [http://2008.igem.org/TUDelft/23_September_2008 23] [http://2008.igem.org/TUDelft/24_September_2008 24] [http://2008.igem.org/TUDelft/25_September_2008 25] [http://2008.igem.org/wiki/index.php?title=TUDelft/26_September_2008&action=edit 26] [http://2008.igem.org/wiki/index.php?title=TUDelft/27_September_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=TUDelft/28_September_2008&action=edit 28]
[http://2008.igem.org/TUDelft/29_September_2008 29] [http://2008.igem.org/TUDelft/30_September_2008 30]
October
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    [http://2008.igem.org/TUDelft/1_October_2008 1] [http://2008.igem.org/TUDelft/2_October_2008 2] [http://2008.igem.org/TUDelft/3_October_2008 3] [http://2008.igem.org/wiki/index.php?title=TUDelft/4_October_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=TUDelft/5_October_2008&action=edit 5]
[http://2008.igem.org/TUDelft/6_October_2008 6] [http://2008.igem.org/TUDelft/7_October_2008 7] [http://2008.igem.org/TUDelft/8_October_2008 8] [http://2008.igem.org/TUDelft/9_October_2008 9] [http://2008.igem.org/TUDelft/10_October_2008 10] [http://2008.igem.org/wiki/index.php?title=TUDelft/11_October_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=TUDelft/12_October_2008&action=edit 12]
[http://2008.igem.org/TUDelft/13_October_2008 13] [http://2008.igem.org/TUDelft/14_October_2008 14] [http://2008.igem.org/TUDelft/15_October_2008 15] [http://2008.igem.org/TUDelft/16_October_2008 16] [http://2008.igem.org/TUDelft/17_October_2008 17] [http://2008.igem.org/wiki/index.php?title=TUDelft/18_October_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=TUDelft/19_October_2008&action=edit 19]
[http://2008.igem.org/TUDelft/20_October_2008 20] [http://2008.igem.org/TUDelft/21_October_2008 21] [http://2008.igem.org/TUDelft/22_October_2008 22] [http://2008.igem.org/TUDelft/23_October_2008 23] [http://2008.igem.org/TUDelft/24_October_2008 24] [http://2008.igem.org/wiki/index.php?title=TUDelft/25_October_2008&action=edit 25] [http://2008.igem.org/wiki/index.php?title=TUDelft/26_October_2008&action=edit 26]
[http://2008.igem.org/wiki/index.php?title=TUDelft/27_October_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=TUDelft/28_October_2008&action=edit 28] [http://2008.igem.org/wiki/index.php?title=TUDelft/29_October_2008&action=edit 29] [http://2008.igem.org/wiki/index.php?title=TUDelft/30_October_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=TUDelft/31_October_2008&action=edit 31]

October 15th

This protocol has been discarded due to the sonicator not being capable of reliably lysing >50 samples.

Cell preparation

  • Pellet 750 ml of grown culture when the culture is around 0.9 by centrifuging 5 min at 10000 rpm in a tabletop centrifuge at 4oC.
  • Resuspend in 1x PBS, keeping the cells on ice.
  • Sonicate the cells by using the sonicator at 2 x 15 seconds, with a 15 second interval, at max amplitude. (decreases as we go in our lab)

Measurements

  • Mix 1 ul of 100X luciferase substrate in 100 ul of assay buffer per sample in the luminometer's reagent tube no. 1
  • Put 20 ul of lysed cells in a white 96 wells plate.
  • Put the prime plate in the luminometer (usually on top)
  • Put the tube with assay buffer under reagent needle no. 1, make sure the tip of the needle is in a position to reach all the assay buffer.
  • Prime the luminometer with 1000 ul assay buffer in the priming plate(make sure the tubing is rinsed before)
  • Measure luciferase activity by:
    • Adding 100 ul of assay buffer to a well
    • Wait 2 seconds
    • Integrate luminescence for 10 seconds.
    • Repeat for every well
    • There is a standard protocol on the computer in which you only have to indicate the wells to be assayed.
  • Purge the tubing (Assay buffer can be stored and frozen for short periods (1 week at most) at -80oC according to the technical manual)
  • Rinse the tubing with 5000 ul of ethanol, and purge it.
  • Rinse the tubing with 5000 ul of H2O, and purge it.
  • The tubing and injector should be clean and empty now.
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