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11 complete 6/23: Seed cells 12 complete 6/24: Transfect hEF1a:eYFP (472 ng/uL) into HEK cells 13 complete 6/26 (4.30pm): Flow Cytometer
  • Determine optimal Lipofectamine LTX volume (24 well)
  • Determine transfection efficiency (quantitatively)

RESULT: Decent transfection efficiency (~60%). Move on to Practice #2.

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

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ATTEMPT #1

36 22 complete 7/3: Seed cells 23 complete 7/4: Transfect hEF1a:eYFP (472 ng/uL) and hEF1a:mKate tagBFP (1176 ng/uL) into HEK cells 24 complete 7/6 (1pm): Flow Cytometer

 

Y1
  • hEF1a:eYFP
(Y+B)1
  • hEF1a:eYFP
  • hEF1a:tagBFP
 ////// ////// ////// -ve control
  • dummy DNA
  • with Lipofectamine
Y2
  • hEF1a:eYFP
(Y+B)2
  • hEF1a:eYFP
  • hEF1a:tagBFP
 ////// ////// ////// -ve control
  • dummy DNA
  • with Lipofectamine
B1
  • hEF1a:tagBFP
(Y+B)3
  • hEF1a:eYFP
  • hEF1a:tagBFP
 ////// ////// ////// -ve/-ve control
  • no DNA
  • no Lipofectamine
B2
  • hEF1a:tagBFP
(Y+B)4
  • hEF1a:eYFP
  • hEF1a:tagBFP
 ////// ////// ////// -ve/-ve control
  • no DNA
  • no Lipofectamine


RESULT: Extremely low transfection efficiency (<10%). Repeat to improve co-transfection results.

 

ATTEMPT #2

22 incomplete 7/_: Seed cells 37 incomplete 7/_: Transfect hEF1a:eYFP and hEF1a:tagBFP into HEK cells 38 incomplete 7/_ (_pm): Flow Cytometer

 

Y1
  • hEF1a:eYFP
(Y+BR)1
  • hEF1a:eYFP
  • hEF1a:mKatetagBFP
//////////////////-ve control
  • dummy DNA
  • with Lipofectamine
Y2
  • hEF1a:eYFP
(Y+RB)2
  • hEF1a:eYFP
  • hEF1a:mKatetagBFP
//////////////////-ve control
  • dummy DNA
  • with Lipofectamine
RB1
  • hEF1a:mKatetagBFP
(Y+RB)3
  • hEF1a:eYFP
  • hEF1a:mKatetagBFP
//////////////////-ve/-ve control
  • no DNA
  • no Lipofectamine
RB2
  • hEF1a:mKatetagBFP
(Y+RB)4
  • hEF1a:eYFP
  • hEF1a:mKatetagBFP
//////////////////-ve/-ve control
  • no DNA
  • no Lipofectamine

 

 

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PRACTICE #3: Fluorescent protein under inducible promoter

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