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TASKWhoDunnitObserved
Split cellsAG, CR, LA, SSAW
Seed platesAG, CR, LA, SSAW
TransfectionAG, LAAC, SS
FACs PrepLACR
Imaging (FloCyto)BTAG, AW, CR, JX, LA

 

Practice Transfections:

Toggle Cloak
CONTROLS:

Cloak
  1. -ve/-ve: no actions performed on cells
  2. -ve: perform protocol, excluding DNA, or with dummy DNA
  3. +ve: tri-color control (perform experiments with YFP, BFP, mKATE separately, and one with all 3: allows for comparison of color/intensity when imaging)


Toggle Cloak
PRACTICE #1: Fluorescent protein under constitutive promoter

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11 complete 6/23: Seed cells 12 complete 6/24: Transfect hEF1a:eYFP into HEK cells 13 complete 6/26 (4.30pm): Flow Cytometer
  • Determine optimal Lipofectamine LTX volume (24 well)
  • Determine transfection efficiency (quantitatively)

Toggle Cloak
PRACTICE #2: Co-transfection (2 fluorescent proteins under constitutive promoter)

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

 

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)

 

 

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

 

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)

 

 

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)