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Experiment #1

Attempt #1

GOAL: Test the strength of L7ae

3 complete 7/14/14: Seed cells 22 complete 7/15/14: Transfect MAV1212-hEF1a-eBFP2 (1084.0 ng/uL), Mutant hEF1a-2xKturn-eGFP (551.4 ng/uL) and Wild Type hEF1a-2xKturn-eGFP (287.0 ng/uL) into HEK cells 0 complete 7/17/14 (12 pm): Flow Cytometer

 

  • DNA Dilution: 5 uL MAV1212-hEF1a-eBFP2, 10 uL Mutant Hef1a-2xKturn-eGFP, 20 uL Wild Type Hef1a-2xKturn-eGFP


24-WELL PLATE TRANSFECTION MAP:

(B)1

  • MAV1212-hEF1a-eBFP2

(B)2

  • MAV1212-hEF1a-eBFP2
////////////////////////

(GM)1

  • Mutant Hef1a-2xKturn-eGFP

(GM)2

  • Mutant Hef1a-2xKturn-eGFP

(GM + B)1

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B)2

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B + L)1

  • Mutant Hef1a-2xKturn-eGFP
  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B + L)2

  • Mutant Hef1a-2xKturn-eGFP
  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT)1

  • Wild Type Hef1a-2xKturn-eGFP

(GWT)2

  • Wild Type Hef1a-2xKturn-eGFP


(GWT + B)1

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B)2

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B + L)1

  • Wild Type Hef1a-2xKturn-eGFP
  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B + L)2

  • Wild Type Hef1a-2xKturn-eGFP
  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

Neg Ctrl

  • Dummy DNA
  • With Lipofectamine


Neg Ctrl

  • Dummy DNA
  • With Lipofectamine


Neg/Neg Ctrl

  • No DNA
  • No Lipofectamin

Neg/Neg Ctrl

  • No DNA
  • No Lipofectamine
////////////

RESULT: Transfection efficiency ~35%. FACS data is available


Attempt #2

2 complete 7/16/14: Seed cells 1 complete 7/17/14: Transfect MAV1212-hEF1a-eBFP2 (1084.0 ng/uL), Mutant hEF1a-2xKturn-eGFP (551.4 ng/uL) and Wild Type hEF1a-2xKturn-eGFP (287.0 ng/uL), MAV1212-hEF1a-L7ae (1686.4 ng/uL) into HEK cells 7 complete 7/19/14 (10 am): Flow Cytometer

 

  • DNA Dilution: 5 uL MAV1212-hEF1a-eBFP2, 10 uL Mutant Hef1a-2xKturn-eGFP, 20 uL Wild Type Hef1a-2xKturn-eGFP, 3 uL MAV1212-hEF1a-L7ae

24-WELL PLATE TRANSFECTION MAP:

(B)1

  • MAV1212-hEF1a-eBFP2

(B)2

  • MAV1212-hEF1a-eBFP2
////////////////////////

(GM)1

  • Mutant Hef1a-2xKturn-eGFP

(GM)2

  • Mutant Hef1a-2xKturn-eGFP

(GM + B)1

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B)2

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B + L)1

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2
  • MAV1212-hEF1a-L7ae

(GM + B + L)2

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2
  • MAV1212-hEFa-L7ae

(GWT)1

  • Wild Type Hef1a-2xKturn-eGFP

(GWT)2

  • Wild Type Hef1a-2xKturn-eGFP


(GWT + B)1

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B)2

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B + L)1

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2
  • MAV1212-hEFa1-L7ae

(GWT + B + L)2

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2
  • MAV1212-hEF1a-L7ae

Neg Ctrl

  • Dummy DNA
  • With Lipofectamine


Neg Ctrl

  • Dummy DNA
  • With Lipofectamine


Neg/Neg Ctrl

  • No DNA
  • No Lipofectamin

Neg/Neg Ctrl

  • No DNA
  • No Lipofectamine
////////////

RESULT: Brief description of experimental outcome (mean transfection efficiency and implications). Link to results page with data analysis


Attempt #3

17 incomplete 7/21/14: Seed cells 18 incomplete 7/22/14: Transfect MAV1212-hEF1a-eBFP2 (1084.0 ng/uL), Mutant hEF1a-2xKturn-eGFP (551.4 ng/uL) and Wild Type hEF1a-2xKturn-eGFP (287.0 ng/uL), MAV1212-hEF1a-L7ae (1686.4 ng/uL), hEF1a-PirB (567.5 ng/uL) into HEK cells 19 incomplete 7/23/14 (10 am): Flow Cytometer

24-WELL PLATE TRANSFECTION MAP:

 

= 0.5 fmoleDummy DNA = ___ fmole

6. (GM + B)2

Mutant Hef1a
  • MAV1212-hEF1a-eBFP2
    • 7M + L
    Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-L7ae
    • 8M )2
    Mutant Hef1a-eBFP2
  • MAV1212-hEFa-L7ae

    • 12. (GWT + B)2

    Wild Type Hef1a
  • MAV1212-hEF1a-eBFP2
    • 13WT
    Wild Type Hef1aeBFP2
  • MAV1212-hEFa1-L7ae
    • 14WT
    Wild Type Hef1aeBFP2
  • MAV1212-hEF1a-L7ae

    • 15. Neg Ctrl

    • Dummy DNA
    • With Lipofectamine
    18

    1.   (B)1

    MAV1212-hEF1a-eBFP2

    (0.11 uL

    = 0.5 fmoleDummy DNA

    )

    hEF1a-PirB (

    ___ uL

    = ___ fmole

    )

    2. (B)2

    MAV1212-hEF1a-eBFP2

    (0.11 uL

    )

    hEF1a-PirB (

    ___ uL

    )

    		////////////////// //////

    3

    		 

    5. (GM WT + B)1

    Mutant Hef1a-2xKturn-eGFP

    2

    MAV1212-hEF1a-eBFP2

    (0.11 uL)

    Wild Type CAG

    -2xKturn-eGFP

    (1.12 uL)

    hEF1a-PirB (___ uL)

    4. (G

    WT + B

    )1

    MAV1212-hEF1a-eBFP2

    (0.11 uL)

    Wild Type CAG-2xKturn-eGFP (1.12 uL)

    hEF1a-PirB (___ uL)

    5. (G

    WT + B + L

    )1

    MAV1212-hEF1a-eBFP2 (0.11 uL)

    Wild Type CAG

    -2xKturn-eGFP (1.12 uL)

    MAV1212-hEF1a

    		   

    -L7ae (0.14 uL)

    hEF1a-PirB (___ uL)

    6. (GWT + B + L)2

    MAV1212-hEF1a-eBFP2 (0.11 uL)

    Wild Type CAG-2xKturn-eGFP (1.12 uL)

    MAV1212-hEF1a-L7ae (0.14 uL)

    hEF1a-PirB (___ uL)

    		////////////

    7. (GM + B)1

    MAV1212-hEF1a-eBFP2 (0.11 uL)

    Multant CAG-2xKturn-eGFP (0.58 uL)

    hEF1a-PirB (___ uL)

    8. (GM + B)1

    11. (GWT + B)1

    Wild Type Hef1a-2xKturn-eGFP

    MAV1212-hEF1a-eBFP2

    (0.11 uL)

    Multant CAG

    -2xKturn-eGFP

    (0.58 uL)

    hEF1a-PirB (___ uL)

    9. (G

    M + B + L)1

    MAV1212-hEF1a-eBFP2 (0.11 uL)

    Multant CAG-2xKturn-eGFP (0.58 uL)

    MAV1212-hEF1a-

    L7ae (0.14 uL)

    hEF1a-PirB (___ uL)

    10. (G

    M + B + L)2

    MAV1212-hEF1a-eBFP2 (0.11 uL)

    Multant CAG-2xKturn-eGFP (0.58 uL)

    MAV1212-hEF1a-

    		  

    L7ae (0.14 uL)

    hEF1a-PirB (___ uL)

    		////////////

    11. Neg Ctrl

    hEF1a-PirB (___ uL)

    With Lipofectamine


    12. Neg Ctrl

    hEF1a-PirB (___ uL)

    With Lipofectamine


    13

    		  16. Neg Ctrl
    • Dummy DNA
    • With Lipofectamine

    17. Neg/Neg Ctrl

    No DNA

    No

    Lipofectamin

    Lipofectamine

    14. Neg/Neg Ctrl

    No DNA

    No Lipofectamine

    		////// //////

    RESULT: Brief description of experimental outcome (mean transfection efficiency and implications). Link to results page with data analysis

    Experiment #2

    GOAL: Test the strength of TAL14

    14 incomplete Day 1: Seed cells 15 incomplete Day 2: Transfect MAV1212-hEF1a-eBFP2 (1084.0 ng/uL), UAS-TAL14-mKate (___ ng/uL), hEF1a-GAL4VP16 (___ ng/uL), hEF1a-TAL14 (___ ng/uL) and UAS-TAL14-eYFP (___ ng/uL) into HEK cells 16 incomplete Day 4: Flow Cytometer

     

    • DNA Dilution: 5 uL MAV1212-hEF1a-eBFP2, ___ uL UAS-TAL14-mKate, ___ uL hEF1a-GAL4VP16, ___ uL hEF1a-TAL14, ___ UAS-TAL14-eYFP


    24-WELL PLATE TRANSFECTION MAP:

    (B)1

      • MAV1212-hEF1a-eBFP2

    (B)2

      • MAV1212-hEF1a-eBFP2
    		////////////

    //////

     

    Neg Ctrl

      • Dummy DNA
      • With Lipofectamine

    (B + Y)1

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-eYFP

    (B + Y)2

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-eYFP

    (B + R)1

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-mKate

    (B + R)2

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-mKate
    		//////

    Neg Ctrl

      • Dummy DNA
      • With Lipofectamine

    (B + Y + GAL)1

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-eYFP
      • hEF1a-Gal4VP16

    (B + Y + GAL)2

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-eYFP
      • hEF1a-Gal4VP16

    (B + R + GAL)1

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-mKate
      • hEF1a-Gal4VP16

    (B + R + GAL)2

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-mKate
      • hEF1a-Gal4VP16
    		//////

    Neg/Neg Ctrl

      • No DNA
      • No Lipofectamin

    (B + Y + GAL)1

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-eYFP
      • hEF1a-Gal4VP16
      • hEF1a-TAL14

    (B + Y + GAL)2

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-eYFP
      • hEF1a-Gal4VP16
      • hEF1a-TAL14

    (B + R + GAL)1

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-mKate
      • hEF1a-Gal4VP16
      • hEF1a-TAL14

    (B + R + GAL)2

      • MAV1212-hEF1a-eBFP2
      • UAS-TAL14-mKate
      • hEF1a-Gal4VP16
      • hEF1a-TAL14
    		//////Neg/Neg Ctrl
      • No DNA
      • No Lipofectamine

    RESULT: Brief description of experimental outcome (mean transfection efficiency and implications). Link to results page with data analysis.

    Experiment #3

    GOAL: Test the sensors in absence of siRNA

    10 incomplete Day 1: Seed cells 11 incomplete Day 2: Transfect Low Sensor miR-144 (___ ng/uL), Low Sensor miR-181c (___ ng/uL), High Sensor miR-30d (___ ng/uL), High Sensor miR-146a (___ ng/uL), High Sensor 125b (___ ng/uL) and High Sensor let-7f (___ ng/uL) into HEK cells 12 incomplete Day 4: (2 pm): Flow Cytometer

     

    • DNA Dilution: ___ uL Low Sensor miR-144, ___ uL Low Sensor miR-181c, ___ uL High Sensor miR-30d, ___ uL High Sensor miR-146a, ___ uL High Sensor 125b, ___ uL High Sensor let-7f


    24-WELL PLATE TRANSFECTION MAP:

    144

      • Low Sensor miR-144

     

      • Low Sensor miR-181c

     

      • High Sensor miR-30d

     

      • High Sensor miR-146a

     

      • High Sensor miR-125b

     

      • High Sensor let-7f

     

      • Low Sensor miR-144

     

      • Low Sensor miR-181c

     

      • High Sensor miR-30d

     

      • High Sensor miR-146a

     

      • High Sensor miR-125b

     

      • High Sensor let-7f

    //////

    //////

    		////////////////////////

    Neg Ctrl

      • Dummy DNA
      • With Lipofectamine


    Neg Ctrl

      • Dummy DNA
      • With Lipofectamine


    Neg/Neg Ctrl

      • No DNA
      • No Lipofectamine

    Neg/Neg Ctrl

      • No DNA
      • No Lipofectamine

     

    //////

     

    //////

    RESULT: Brief description of experimental outcome (mean transfection efficiency and implications). Link to results page with data analysis.

    Experiment #4

    GOAL: Test the sensors in the presence of siRNA

    13 incomplete Day 1: Seed cells 9 incomplete Day 2: Transfect Low Sensor miR-144 (___ ng/uL), Low Sensor miR-181c (___ ng/uL), High Sensor miR-30d (___ ng/uL), High Sensor miR-146a (___ ng/uL), High Sensor 125b (___ ng/uL), High Sensor let-7f (___ ng/uL), Mature 144 (___ ng/uL), Mature 181c (___ ng/uL), Mature 30d (___ ng/uL), Mature 146a (___ ng/uL), Mature 125b (___ ng/uL) and Mature let-7f (___ ng/uL) into HEK cells 20 incomplete Day 4: (2 pm): Flow Cytometer

     

    • DNA Dilution: ___ uL Low Sensor miR-144, ___ uL Low Sensor miR-181c, ___ uL High Sensor miR-30d, ___ uL High Sensor miR-146a, ___ uL High Sensor 125b, ___ uL High Sensor let-7f , ___ uL Mature 144, ___ uL Mature 181c, ___ uL Mature 30d, ___ uL Mature 146a, ___ uL Mature 125b and ___ uL Mature let-7f


    24-WELL PLATE TRANSFECTION MAP:

    144

      • Low Sensor miR-144
      • Mature 144

     

      • Low Sensor miR-181c
      • Mature 181c

     

      • High Sensor miR-30d
      • Mature 30d

     

      • High Sensor miR-146a
      • Mature 146a

     

      • High Sensor miR-125b
      • Mature 125b

     

      • High Sensor let-7f
      • Mature let-7f

     

      • Low Sensor miR-144
      • Mature 144

     

      • Low Sensor miR-181c
      • Mature 181c

     

      • High Sensor miR-30d
      • Mature 30d

     

      • High Sensor miR-146a
      • Mature 146a

     

      • High Sensor miR-125b
      • Mature 125b

     

      • High Sensor let-7f
      • Mature let-7f

    //////

    //////

    		////////////////////////

    Neg Ctrl

      • Dummy DNA
      • With Lipofectamine


    Neg Ctrl

      • Dummy DNA
      • With Lipofectamine


    Neg/Neg Ctrl

      • No DNA
      • No Lipofectamine

    Neg/Neg Ctrl

      • No DNA
      • No Lipofectamine

     

    //////

     

    //////

    RESULT: Brief description of experimental outcome (mean transfection efficiency and implications). Link to results page with data analysis.

    ...