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:


CONTROLS:

  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)


PRACTICE #1: Fluorescent protein under constitutive promoter

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

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

  • 7/2: Seed cells
  • 7/3: Transfect hEF1a:eYFP and hEF1a:mKate into HEK cells
  • 7/5 (__pm): Flow Cytometer

PLATE:

 Y2
  • _uL complete media
  • _uL hEF1a:eYFP
  • _uL Lipofectamine LTX
/////////

-ve control

  • dummy DNA
 Y4
  • _uL complete media
  • _uL hEF1a:eYFP
  • _uL Lipofectamine LTX
/////////

-ve control

  • dummy DNA
 R2
  • _uL complete media
  • _uL hEF1a:mKate
  • _uL Lipofectamine LTX
/////////

-ve/-ve control

  • cells (no action)
 R4
  • _uL complete media
  • _uL hEF1a:mKate
  • _uL Lipofectamine LTX
 		/////////

-ve/-ve control

  • cells (no action)


 

 

PRACTICE #3: Fluorescent protein under inducible promoter

  • 7/_: Seed cells
  • 7/_: Transfect TRE:mKate, hEF1a:rtTA and hEF1a:eYFP (transfection marker) into HEK cells
  • 7/_: Add DOX
  • 7/_ (__pm): Flow Cytometer


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

  • 7/_: Seed cells
  • 7/_: Transfect hEF1a:eYFP, TRE:mKate and hEF1a:rtTA** into HEK cells
  • 7/_: Add DOX
  • 7/_ (__pm): Flow Cytometer