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Cloak
14 incomplete 7/_: Seed cells 15 incomplete 7/_: Transfect TRE:mKate, hEF1a:rtTA and hEF1a:eYFP (transfection marker) into HEK cells 16 incomplete 7/_: Add DOX 18 incomplete 7/_ (__pm): Flow Cytometer

 

  • DNA Dilution: __uL hEF1a:eYFP; __uL hEF1a:rtTA; __uL TRE:mKate
  • (D=x): add _:_:_
    1. __uL hEF1a:eYFP-lipid complex
    2. __uL hEF1a:rtTA-lipid complex
    3. __uL TRE:mKate-lipid complex
+ve control
  • hEF1a:eYFP
+ve control
  • hEF1a:eYFP
-ve control
  • dummy DNA
  • with Lipofectamine
-ve control
  • dummy DNA
  • with Lipofectamine
-ve/-ve control
  • no DNA
  • no Lipofectamine
-ve/-ve control
  • no DNA
  • no Lipofectamine
////////////////////////////////////
(D=0)1
  • no DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
(D=0.)2
  • 0.2uM no DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=10.2
  • 0.2uM
  • 1uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=1.5
  • 1.5uM 1uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=31.5
  • 1.5uM
  • 3uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=43
  • 4uM 3uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
(D=0)24
  • no 4uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=6
  • 6uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=7.5
  • 7.5uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=10
  • 10uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=17
  • 17uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=30
  • 30uM DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate

RESULTS:

Toggle Cloak
PRACTICE #4**: Co-transfection (2 fluorescent proteins under constitutive & inducible promoters)

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