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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: Transfection efficiency ~35%. FACS data is available


Attempt #3

17 complete 7/21/14: Seed cells 18 incomplete 7/22/14: Transfect MAV1212-hEF1a-eBFP2, Mutant hEF1a-2xKturn-eGFP, Wild Type hEF1a-2xKturn-eGFP, MAV1212-hEF1a-L7ae, hEF1a-PirB (dummy DNA) all at ~500 ng/uL into HEK cells 19 incomplete 7/23/14 (10 am): Flow Cytometer
  • DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), Wild Type CAG-2xKturn-eGFP (0.96 uL), Mutant CAG-2xKturn-eGFP (0.96 uL), MAV1212-hEF1a-L7ae (0.71 uL), hEF1a-PirB (to 1 ug)

24-WELL PLATE TRANSFECTION MAP:

1. (B)1

MAV1212-hEF1a-eBFP2 (1.0 fmole)

hEF1a-PirB (1.5 fmole = 1.60 uL)

2. (B)2

MAV1212-hEF1a-eBFP2 (1.0 fmole)

hEF1a-PirB (1.5 fmole = 1.60 uL)

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

3. (GWT + B)2

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

4. (GWT + B)1

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

5. (GWT + B + L)1

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

6. (GWT + B + L)2

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Wild Type CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

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

7. (GM + B)1

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

8. (GM + B)1

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

9. (GM + B + L)1

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

10. (GM + B + L)2

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

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

11. Neg Ctrl

hEF1a-PirB (3.2 fmoles = 2.05 uL)

With Lipofectamine


12. Neg Ctrl

hEF1a-PirB (3.2 fmoles = 2.05 uL)

With Lipofectamine


13. Neg/Neg Ctrl

No DNA

No Lipofectamine

14. Neg/Neg Ctrl

No DNA

No Lipofectamine

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

RESULT: Transfection efficiency ___%. FACS data is available

 

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 + (B +

1. (B)1

MAV1212-hEF1a-eBFP2

2. (B)2

MAV1212-hEF1a-eBFP2

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

//////

 

3. Neg Ctrl

Dummy DNA

With Lipofectamine

(B + Y)1

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

(B + Y)2

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

4. (B + R)1

MAV1212-hEF1a-eBFP2

UAS-TAL14-mKate

5. (B + R)2

MAV1212-hEF1a-eBFP2

UAS-TAL14-mKate

 

 //////

6. Neg Ctrl

Dummy DNA

With Lipofectamine

7. (B +

Y

R + GAL)1

MAV1212-hEF1a-eBFP2

UAS-TAL14-

eYFP

mKate

hEF1a-Gal4VP16

8. (B +

Y

R + GAL)2

MAV1212-hEF1a-eBFP2

UAS-TAL14-

eYFP

mKate

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

 

 //////

9. 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

Lipofectamine

10. (B +

R + GAL)1

MAV1212-hEF1a-eBFP2

UAS-TAL14-mKate

hEF1a-Gal4VP16

hEF1a-TAL14

11. (B + R + GAL)2

MAV1212-hEF1a-eBFP2

UAS-TAL14-mKate

hEF1a-Gal4VP16

hEF1a-TAL14

 

 //////12. Neg/Neg Ctrl

No DNA

No Lipofectamine

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.

...