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TASKWhoDunnitObserved
Split cellsAG, CR, GB, JA, LA, SSAW
Seed platesAG, CR, GB, LA, SSAW
TransfectionAG, GB, LAAC, SS

...

FACS PrepAG, LACR
Imaging (FloCyto)BTAG, AW, CR, JX, LA

 

Practice Transfections:

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CONTROLS

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Cloak

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  1. +ve: single color transfection (eYFP, eBFP or mKate, as appropriate)
  2. +ve/+ve

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  1. : tri-color control (

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  1. eYFP, eBFP and mKate co-transfected: allows for comparison of color/intensity when imaging)

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  2. -ve: perform protocol, excluding DNA, or with dummy DNA
  3. -ve/-ve: no actions performed on cells

...

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PRACTICE #1: Fluorescent protein under constitutive promoter

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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

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36 complete 7/3: Seed cells 23 complete 7/4: Transfect hEF1a:eYFP (472 ng/uL) and hEF1a:

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tagBFP (1176 ng/uL) into HEK cells 24 complete 7/6 (1pm): Flow Cytometer

 

  • DNA Dilution: 10uL hEF1a:eYFP; 5uL hEF1a:tagBFP
  • (Y+B)x: add 1:1 (25uL hEF1a:eYFP/tagBFP-lipid complex)
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:

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  • tagBFP
(Y+

...

B)3
  • hEF1a:eYFP
  • hEF1a:

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  • 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 complete 7/9: Seed cells 37 complete 7/10: Transfect hEF1a:mKate (621 ng/uL) and hEF1a:eBFP (405 ng/uL) into HEK cells 38 complete 7/12 (1:30pm): Flow Cytometer

 

  • DNA Dilution: 8.1uL (10 uL) hEF1a:mKate; 12.3uL (15 uL) hEF1a:eBFP
  • (R+B)x: add 1:1 (25uL hEF1a:mKate/eBFP-lipid complex)
R1
  • hEF1a:mKate
(R+B)1
  • hEF1a:mKate
  • hEF1a:eBFP
//////////////////-ve control
  • dummy DNA
  • with Lipofectamine
R2
  • hEF1a:mKate
(

...

R+B)2
  • hEF1a:mKate
  • hEF1a:eBFP
//////////////////-ve control
  • dummy DNA
  • with Lipofectamine
B1
  • hEF1a:eBFP
(R+B)3
  • hEF1a:mKate
  • hEF1a:eBFP
//////////////////-ve/-ve control
  • no DNA
  • no Lipofectamine
B2
  • hEF1a:eBFP
(R+B)4
  • hEF1a:

...

  • mKate
  • hEF1a:

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  • eBFP
//////////////////-ve/-ve control
  • no DNA
  • no Lipofectamine

RESULT: Efficiency ~20%

 

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PRACTICE #3: Fluorescent protein under inducible promoter (DOX ladder)

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14

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complete 7/

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10: Seed cells15

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complete 7/

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11: Transfect TRE:mKate (431 ng/uL), hEF1a:rtTA (169 ng/uL) and hEF1a:eYFP (1045 ng/uL - transfection marker) into HEK cells 16

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complete 7/

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12: Add DOX18

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complete 7/

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13 (1:30pm): Flow Cytometer

 

  • DNA Dilution: 4.8uL hEF1a:eYFP; 29.6uL hEF1a:rtTA; 11.6uL TRE:mKate
  • Conc. of DOX: 1mg/uL
  • (D=x): add 2:1:2
    1. 20uL hEF1a:eYFP-lipid complex
    2. 10uL hEF1a:rtTA-lipid complex
    3. 20uL TRE:mKate-lipid complex

 

+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
////////////////////////////////////
(D=0)1
  • no DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
(D=0

...

)2

...

  • no DOX
  • hEF1a:eYFP
  • hEF1a:rtTA
  • TRE:mKate
D=1

    ...

    • 1mg DOX
    • hEF1a:eYFP
    • hEF1a:rtTA
    • TRE:mKate
    		D=

    ...

    2(*3)
    • 2mg DOX
    • hEF1a:eYFP
    • hEF1a:rtTA
    • TRE:mKate
    		D=3(*5)

      ...

      • 3mg DOX
      • hEF1a:eYFP
      • hEF1a:rtTA
      • TRE:mKate
      		D=4(*10)

        ...

        • 4mg DOX
        • hEF1a:eYFP
        • hEF1a:rtTA
        • TRE:mKate

        ...

        D=

        ...

        5(*2)

        ...

          ...

          • 5mg DOX
          • hEF1a:eYFP
          • hEF1a:rtTA
          • TRE:mKate
          		D=6(*4)

          ...

          • 6mg DOX
          • hEF1a:eYFP
          • hEF1a:rtTA
          • TRE:mKate
          		D=7

          ...

          (*6)
          • 7mg

          ...

          • DOX
          • hEF1a:eYFP
          • hEF1a:rtTA
          • TRE:mKate
          		D=

          ...

          8(*7)
          • 8mg DOX
          • hEF1a:eYFP
          • hEF1a:rtTA
          • TRE:mKate
          		D=

          ...

          9(*8)
          • 9mg

          ...

          • DOX
          • hEF1a:eYFP
          • hEF1a:rtTA
          • TRE:mKate
          		D=

          ...

          10(*9)
          • 10mg

          ...

          • DOX
          • hEF1a:eYFP
          • hEF1a:rtTA
          • TRE:mKate

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

          ...

          RESULTS: Efficiency >50%


          SUBGROUP-SPECIFIC PRACTICE EXPERIMENTS

          Inducible and Constitutive Cascades

          Image Added