Haynes Lab:Notebook/Short Projects/2015/03/01: Difference between revisions

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'''Passaging - KAH126'''
'''Passaging - KAH126'''


* Protocol:  
* Protocol: [http://openwetware.org/wiki/Haynes:SplittingCells Splitting Cells]
* Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S
* Use 90-100% confluent '''T-75'''
* Use 90-100% confluent '''T-75'''
* Make 1 new T-75 at a 1:10 dilution
* Passage number: 2


----
----
'''6-well plate - KAH126'''
'''6-well plate - KAH126'''


* Start with 90-100% confluent '''T-75''' = 10E6/10 mL = 1.0E6/1 mL
* Use same resuspension from old flask in '''Passaging - KAH126'''
*  
* Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration
* Desired cells/ well in new 6-well plate = '''2.5E5 (250k) cells in 4.0 mL medium'''
* Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells  = '''0.25 mL stock cell resuspension per well'''
* Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = '''3.75 mL fresh medium per well'''
* Make a batch of diluted cells for new 6-well plate:
** Into a sterile 50 mL conical add...
** (6 + 1 ''pipetting error'') x 0.25 mL stock cells = 1.75 mL stock cells
** (6 + 1 ''pipetting error'') x 3.75 mL fresh medium = 26.25 mL fresh medium




Revision as of 16:55, 25 March 2015

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Karmella Haynes - 03/01/15

  • Passaging - KAH126, split 1:10
  • 6-well plate - KAH126
  • Dox induction - KAH126

Passaging - KAH126

  • Protocol: Splitting Cells
  • Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S
  • Use 90-100% confluent T-75
  • Make 1 new T-75 at a 1:10 dilution
  • Passage number: 2

6-well plate - KAH126

  • Use same resuspension from old flask in Passaging - KAH126
  • Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration
  • Desired cells/ well in new 6-well plate = 2.5E5 (250k) cells in 4.0 mL medium
  • Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells = 0.25 mL stock cell resuspension per well
  • Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = 3.75 mL fresh medium per well
  • Make a batch of diluted cells for new 6-well plate:
    • Into a sterile 50 mL conical add...
    • (6 + 1 pipetting error) x 0.25 mL stock cells = 1.75 mL stock cells
    • (6 + 1 pipetting error) x 3.75 mL fresh medium = 26.25 mL fresh medium



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