Haynes Lab:Notebook/Jan/2015/04/21: Difference between revisions

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==Finishing Creating a Stable Cell Line for BD006/BD004 U2OS/Splitting New U2OS Cells==
==Finishing Creating a Stable Cell Line for BD006/BD004 U2OS/Splitting New U2OS Cells==
* Yesterday we seeded the U2OS cells transfected with BD004 and BD006 into 10 cm plates. Protocol for today:
* Yesterday we seeded the U2OS cells transfected with BD004 and BD006 into 10 cm plates. Protocol for today:
** The next day (18-24 hours later), add selective medium (based on the resistance marker for your plasmid). In this case, McCoy's 5A w/FBS and P/S with puromycin at a concentration of 1.0 ug/mL. Refer to Media Formulas protocol page for more information.
** The next day (18-24 hours later), aspirate off old medium and add selective medium (based on the resistance marker for your plasmid). In this case, McCoy's 5A w/FBS and P/S with puromycin at a concentration of 1.0 ug/mL. Refer to Media Formulas protocol page for more information.
** Grow the cells for 1 - 2 weeks until isolated colonies (about 100 cells per colony) appear. Transfer single colonies into 24-well plates. Grow the cells until they reach 100% confluency. Expand the cells in larger growth vessels (e.g., 6-well plates or 10 cm plates) for further testing.
** Grow the cells for 1 - 2 weeks until isolated colonies (about 100 cells per colony) appear. Transfer single colonies into 24-well plates. Grow the cells until they reach 100% confluency. Expand the cells in larger growth vessels (e.g., 6-well plates or 10 cm plates) for further testing.


Revision as of 14:54, 21 April 2015

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Finishing Creating a Stable Cell Line for BD006/BD004 U2OS/Splitting New U2OS Cells

  • Yesterday we seeded the U2OS cells transfected with BD004 and BD006 into 10 cm plates. Protocol for today:
    • The next day (18-24 hours later), aspirate off old medium and add selective medium (based on the resistance marker for your plasmid). In this case, McCoy's 5A w/FBS and P/S with puromycin at a concentration of 1.0 ug/mL. Refer to Media Formulas protocol page for more information.
    • Grow the cells for 1 - 2 weeks until isolated colonies (about 100 cells per colony) appear. Transfer single colonies into 24-well plates. Grow the cells until they reach 100% confluency. Expand the cells in larger growth vessels (e.g., 6-well plates or 10 cm plates) for further testing.


  • Splitting for Today
  • 1 T-75 Flask (1-9)
  • 1 6-Well Plate (1-7)


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