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Cloak
22 incomplete 7/2: Seed cells 23 incomplete 7/3: Transfect hEF1a:eYFP and hEF1a:mKate into HEK cells 24 incomplete 7/5 (__pm): Flow Cytometer

 

cells ( action)cells ( action)
Y1
  • 50uL DNA-lipid complex
  • _uL hEF1a:eYFP
  • _uL Lipofectamine LTX
(Y+R)1
  • 50uL DNA-lipid complex
  • hEF1a:eYFP
  • _uL hEF1a:mKate
  • _uL Lipofectamine LTX
 ////// ////// ////// -ve control
  • dummy DNA
  • with Lipofectamine
Y2
    50uL DNA-lipid complex
  • _uL hEF1a:eYFP
  • _uL Lipofectamine LTX
(Y+R)2
  • 50uL Opti-MEM medium
  • hEF1a:eYFP
  • _uL hEF1a:mKate_uL Lipofectamine LTX
 ////// ////// ////// -ve control
  • dummy DNA
  • with Lipofectamine
R1
  • 50uL DNA-lipid complex
  • _uL hEF1a:mKate
  • _uL Lipofectamine LTX
(Y+R)3
  • 50uL Opti-MEM medium
  • hEF1a:eYFP
  • _uL hEF1a:mKate_uL Lipofectamine LTX
 ////// ////// ////// -ve/-ve control
  • no DNA
  • no
  • Lipofectamine
R2
  • 50uL DNA-lipid complex
  • _uL hEF1a:mKate_uL Lipofectamine LTX
(Y+R)4
  • 50uL Opti-MEM medium
  • hEF1a:eYFP
  • _uL hEF1a:mKate_uL Lipofectamine LTX
 ////// ////// ////// -ve/-ve control
    • no
    • DNA
     
    • no Lipofectamine

     

    Toggle Cloak
    PRACTICE #3: Fluorescent protein under inducible promoter

    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

     

    (Y+R)1
  • _uL complete media
  • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    +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
    		Y1
    • _uL complete media
    • _uL hEF1a:eYFP
    • _uL Lipofectamine LTX
    //////////////////

    -ve control

    • dummy DNA
    		Y2
    • _uL complete media
    • _uL hEF1a:eYFP
    • _uL Lipofectamine LTX
    		(Y+R)2
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		//////////////////

    -ve control

    • dummy DNA
    R1
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		(Y+R)3
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		//////////// //////

    -ve/-ve control

    • cells (no action)
    R2
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		(Y+R)4
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		//////////// //////

    -ve/-ve control

    • cells (no action)

     

     
    (D=0)1
    • no DOX
    • hEF1a:eYFP
    • hEF1a:rtTA
    • TRE:mKate
    		D=0.2
    • 0.2uM DOX
    • hEF1a:eYFP
    • hEF1a:rtTA
    • TRE:mKate
    		D=1
    • 1uM DOX
    • hEF1a:eYFP
    • hEF1a:rtTA
    • TRE:mKate
    		D=1.5
    • 1.5uM DOX
    • hEF1a:eYFP
    • hEF1a:rtTA
    • TRE:mKate
    		D=3
    • 3uM DOX
    • hEF1a:eYFP
    • hEF1a:rtTA
    • TRE:mKate
    		D=4
    • 4uM DOX
    • hEF1a:eYFP
    • hEF1a:rtTA
    • TRE:mKate
    (D=0)2
    • no 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

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

    Cloak
    29 incomplete 7/_: Seed cells 30 incomplete 7/_: Transfect hEF1a:eYFP, TRE:mKate and hEF1a:rtTA** into HEK cells 31 incomplete 7/_: Add DOX 32 incomplete 7/_ (__pm): Flow Cytometer

     

    Y1
    • _uL complete media
    • _uL hEF1a:eYFP
    • _uL Lipofectamine LTX
    		(Y+R)1
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		 ////// ////////////

    -ve control

    • dummy DNA
    Y2
    • _uL complete media
    • _uL hEF1a:eYFP
    • _uL Lipofectamine LTX
    		(Y+R)2
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		//////////////////

    -ve control

    • dummy DNA
    R1
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		(Y+R)3
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		//////////////////

    -ve/-ve control

    • cells (no action)
    R2
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		(Y+R)4
    • _uL complete media
    • _uL hEF1a:mKate
    • _uL Lipofectamine LTX
    		//////////////////

    -ve/-ve control

    • cells (no action)