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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)

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PRACTICE #2: Fluorescent protein under inducible promoterCo-transfection (2 fluorescent proteins under constitutive promoter)

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14 22 incomplete 7/2: Seed cells 15 23 incomplete 7/3: Transfect TREhEF1a:eYFP and hEF1a:mKate /TRE:eYFP into HEK cells and hEF1a:rtTA and hEF1a:_FP 16 incomplete 7/4: Add DOX 18 incomplete 7/5 (__pm): Flow Cytometer 24 incomplete 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)


 

 

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PRACTICE #3: Co-transfection (2 fluorescent proteins under constitutive promoter)Fluorescent protein under inducible promoter

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22 14 incomplete 7/_: Seed cells 23 15 incomplete 7/_: Transfect TRE:mKate, hEF1a:____ rtTA and hEF1a:____ eYFP (transfection marker) into HEK cells 24 16 incomplete 7/_: Add DOX 18 incomplete 7/_ (__pm): Flow Cytometer


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

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