User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/17

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06/17/2013

Prepare Transfected Cells for Hygromycine Treatment

Transfer Transfected Cells from 6 well plate to 10cm cell culture dishes. Target cell concentrations are: 1x106, 1x105, 1x104, 1x103


1x106 Monolayer 100% confluent cells are attached to the bottom of each well

  • Add 2mL Trypsin to each of the wells 1 and 2 for 4 minutes.
  • Add 3.5mL U2OS media (p/s) to neutralize Trypsin.
  • Add 6mL U2OS media (p/s) to the 4ml of well 1.
  • Transfer all 10mL of well 1 to 10cm plate to have 1x106.
  • Add 16mL U2OS media (p/s) to the 4mL of well 2.
  • Take 2mL of cells, dilute with 8mL of U2OS media (p/s) and transfer to 10cm plate 1x105.
  • Take 1mL of cells in well 2, dilute with 9mL of U2OS media (p/s) and transfer to 10cm plate 1x104.
  • Take 0.5mL of cells in well 2, dilute in 9.5mL of U2OS media (p/s) and transfer to 10cm plate 1x103.

Keep cells in the incubator to reach 90% confluency.

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