User:David K. Barclay/Notebook/Controlling Pancreas Cell Fate Using Transcription Factors/2014/03/31

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TC1-Alpha cells transfection redo

  • TC1-Alpha cells transfected 10 days ago were dead due to overgrowth. Transfection needs to be redone.
  • Two T-75 flasks(10mL at 100% confluence) of the TC1-Alpha Cells

Transfection (6 well)

  • T-75(1) container used
  • Each well done 1/10, 1mL detached cells to 3mL DMEM.
  • 6 wells= 6/10 of one T-75 used.

Splitting Cells

  • Rest of T75(1) container used.
  • Split cells 1/5, 2mL detached cells to 8mL DMEM.
  • Rest of T75(1) container disposed
  • Protocol used for these two functions was the Splitting Cell protocol

Freezing Cells

  • Concentration: 90% FBS, 10% DMSO
  • T75(2) flask used, detach cells according to Splitting Cell protocol
  • Transfer cells to spin column and spin down
  • Aspirate fluid, leaving enough to cover top of pellet, re-suspend pellet
  • Add 5mL Freezing Serum
  • Remix cells in Freezing serum
  • 1mL into tubes, 5 tubes created for freezing