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TASKWhoDunnitObserved
Split cellsAG, CR, GB, JA, LA, SSAW
Seed platesAG, CR, GB, LA, SSAW
Transfection

...

AG, GB, LAAC, SS
FACS PrepAG, LACR
Imaging (FloCyto)BTAG, AW, CR, JX, LA

 

Practice Transfections:

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CONTROLS

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  1. +ve: single color transfection (eYFP, eBFP or mKate, as appropriate)
  2. +ve/+ve: tri-color control (eYFP, eBFP and mKate co-transfected: allows for comparison of color/intensity when imaging)
  3. -ve: perform protocol, excluding DNA, or with dummy DNA
  4. -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

    Practice Transfection:

    CONTROLS:

    1. -ve/-ve: 
    2. -ve:
    3. +ve:

     

     

    • 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 complete 7/3: Seed cells 23 complete 7/4: Transfect hEF1a:eYFP (472 ng/uL) and hEF1a: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: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 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: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 complete 7/10: Seed cells 15 complete 7/11: Transfect TRE:mKate (431 ng/uL), hEF1a:rtTA (169 ng/uL) and hEF1a:eYFP (1045 ng/uL - transfection marker) into HEK cells 16 complete 7/12: Add DOX 18 complete 7/13 (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

    RESULTS: Efficiency >50%


    SUBGROUP-SPECIFIC PRACTICE EXPERIMENTS

    Inducible and Constitutive Cascades

    Image Added

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