Haynes Lab:Notebook/Short Projects/2014/09/11: Difference between revisions

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==Entry title==
==9/11/14 - Jonah Thomas==
* Insert content here...
* Cell culture - check cells, add dox to dox+ cells
* Plan transfections - measure concentration of plasmid DNA
 
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'''Cell culture'''
* Checked cells. Cells are attached, about 50 - 60 % confluent. Will do Transfections on Friday.
* Added 1 mg/mL doxycycline (dox) to a final concentration of 1 μg/mL to dox+ 6-well plate and T-75 flask
** Added 4 μL dox directly to wells in 6-well plate
** Transferred 2 mL medium from flask to 15 mL conical, added 10 μL dox, added 2 mL back to flask with sterile pipette
 
 
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'''Plan Transfections'''
* Transription factor-expressing plasmid is KAH60/pcVN: https://benchling.com/hayneslab/mammalian-constructs/WEh-kah60pcvn/
* There is plenty of plasmid stock (~100 μL) from Dr. Haynes' Harvard box (H4)
* KAH60/pcVN concentration written on tube as ~450 ng/μL
* Need to re-measure to be certain
 
 
Measurement (Biotek machine - Take 3 plate)
* OD260 = 0.384
* 260/280 = 1.88
* '''ng/μL = 384.2'''
 
 
Strategy for transfection: add Lipo/DNA to 6 wells total...
* Will need 2000 ng KAH60/pcVN DNA per transfecttion
* Plate 1 - no dox, transfect 3 wells only
* Plate 2 - dox+, transfect 3 wells only




Revision as of 13:43, 11 September 2014

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9/11/14 - Jonah Thomas

  • Cell culture - check cells, add dox to dox+ cells
  • Plan transfections - measure concentration of plasmid DNA

Cell culture

  • Checked cells. Cells are attached, about 50 - 60 % confluent. Will do Transfections on Friday.
  • Added 1 mg/mL doxycycline (dox) to a final concentration of 1 μg/mL to dox+ 6-well plate and T-75 flask
    • Added 4 μL dox directly to wells in 6-well plate
    • Transferred 2 mL medium from flask to 15 mL conical, added 10 μL dox, added 2 mL back to flask with sterile pipette


Plan Transfections


Measurement (Biotek machine - Take 3 plate)

  • OD260 = 0.384
  • 260/280 = 1.88
  • ng/μL = 384.2


Strategy for transfection: add Lipo/DNA to 6 wells total...

  • Will need 2000 ng KAH60/pcVN DNA per transfecttion
  • Plate 1 - no dox, transfect 3 wells only
  • Plate 2 - dox+, transfect 3 wells only



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