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Toggle Cloak
Antibody Detection

Cloak

 

Toggle Cloak
Receptor Detection

Cloak

 

Toggle Cloak
miRNA Detection

Transfection 1: Transfecting TRE:Syk-TEVp for Western Blot to compare the levels of endogenous vs exogenous Syk and to compare levels of expression under different amounts of dox induction.

Concentrations of DNA:

Dummy1230 ng/ul
hEF1a:eYFP1045 ng/ul
hEF1a:rtTa169.4 ng/ul
TRE:Syk-TEVp871 ng/ul

Volumes of DNA added:

Well 3Well 4Wells 7-12
Dummy 0.81 ul

Dummy 0.49 ul

hEF1a:eYFP 0.49

hEF1a:eYFP 0.38 ul

hEF1a:rtTA 1.18 ul

TRE:Syk-TEVp 0.46 ul

Well 1

HEK293

Well 2

EMPTY

(Would have been B cells)

Well 3

HEK293

Dummy DNA 1000 ng

Well 4

HEK293

hEF1a:eYFP 400 ng

Dummy DNA 600 ng

Well 5

EMPTY

Well 6

EMPTY

Well 7

HEK293

hEF1a:eYFP 400 ng

hEF1a: rtTA 200 ng

TRE: Syk-TEVp 400 ng

0 nM Dox

Well 8

HEK293

hEF1a:eYFP 400 ng

hEF1a: rtTA 200 ng

TRE: Syk-TEVp 400 ng

1 nM Dox

Well 9

HEK293

hEF1a:eYFP 400 ng

hEF1a: rtTA 200 ng

TRE: Syk-TEVp 400 ng

10 nM Dox

Well 10

HEK293

hEF1a:eYFP 400 ng

hEF1a: rtTA 200 ng

TRE: Syk-TEVp 400 ng

100 nM Dox

Well 11

HEK293

hEF1a:eYFP 400 ng

hEF1a: rtTA 200 ng

TRE: Syk-TEVp 400 ng

1000 nM Dox

Well 12

HEK293

hEF1a:eYFP 400 ng

hEF1a: rtTA 200 ng

TRE: Syk-TEVp 400 ng

2000 nM Dox

      
      

Toggle Cloak
Receptor Detection

Cloak

 

Toggle Cloak
miRNA Detection

 

 

(GWT)2

  • Wild Type Hef1a-2xKturn-eGFP

Neg Ctrl

  • Dummy DNA
  • With Lipofectamine

Neg Ctrl

  • Dummy DNA
  • With Lipofectamine

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

Attempt #2

2 complete 7/16/14: Seed cells 1 incomplete 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 0 complete 7 incomplete 7/1917/14 (2 12 pm): 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


24-24-WELL PLATE TRANSFECTION MAP:

Cloak

L7ae Repression

Attempt #1

GOAL: Test the strength of L7ae

3 complete 7/14/14: Seed cells 22 complete 7/15/14: Transfect
Cloak
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 incomplete 7/17/14 (2 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 + 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/Neg Ctrl

  • No DNA
  • No Lipofectamin

Neg/Neg Ctrl

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

(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
  • hEF1a-L7ae

(GM + B + L)2

  • Mutant Hef1a-2xKturn-eGFP
  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2
  • 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
  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2hEFa1-L7ae

(GWT + B + L)2

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

Experiment #2

Inconclusive. No L7ae was added.


Attempt #2

2 complete 7/16/14: Seed cells 1 complete 7/17/14

GOAL: Test the strength of TAL14

14 incomplete Day 1: Seed cells 15 incomplete Day 2: Transfect MAV1212-hEF1a-eBFP2 (1084.0 ng/uL), UASMutant hEF1a-TAL142xKturn-mKate (___ eGFP (551.4 ng/uL) , and Wild Type hEF1a-GAL4VP16 (___ 2xKturn-eGFP (287.0 ng/uL), MAV1212-hEF1a-TAL14 (___ ng/uL) and UAS-TAL14-eYFP (___ L7ae (1686.4 ng/uL) into HEK cells 16 incomplete Day 4: Flow 7 complete 7/19/14 (10 am): Flow Cytometer

 

  • DNA Dilution: 5 uL MAV1212-hEF1a-eBFP2, ___ uL UAS-TAL14-mKate, ___ uL hEF1a-GAL4VP16, ___ uL hEF1a-TAL14, ___ UAS-TAL14-eYFP10 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
//////////////////

 

Neg Ctrl

    • Dummy DNA
    • With Lipofectamine
//////

(GM

(B + Y

)1

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

(GM)2

  • Mutant Hef1a-2xKturn-eGFP

(GM + B)1

  • Mutant Hef1a-2xKturn-eGFP
  • UAS-TAL14-eYFP
    • (B + Y)2
    • MAV1212-hEF1a-eBFP2
  • UAS-TAL14-eYFP

    • (B + R)1

    (GM + B)2

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

    (GM + B + L)1

    • Mutant Hef1a-2xKturn-eGFP
    • MAV1212-hEF1a-eBFP2
    UAS
    • MAV1212-
    TAL14
    • hEF1a-
    mKate
    • L7ae

    (GM + B +

    R

    L)2

    • Mutant Hef1a-2xKturn-eGFP
    • MAV1212-hEF1a-eBFP2
    UAS
    • MAV1212-
    TAL14
    • hEFa-
    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

    • 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
    UAS
    • MAV1212-
    TAL14-mKatehEF1a-Gal4VP16
    • hEFa1-L7ae

    (GWT + B +

    R + GAL

    L)2

    • Wild Type Hef1a-2xKturn-eGFP
    • MAV1212-hEF1a-eBFP2
    UAS
    • MAV1212-
    TAL14-mKate
  • hEF1a-Gal4VP16
    • hEF1a-L7ae

    Neg Ctrl

    • Dummy DNA
    • With Lipofectamine


    Neg Ctrl

    • Dummy DNA
    • With Lipofectamine
    //////


    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

    Neg/Neg Ctrl

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


    RESULT: Inconclusive: Too much DNA in each well.


    Attempt #3

    17 complete 7/24/14: Seed cells 18 complete 7/25/14: Transfect 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, 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 complete 7/27/14 (10 am): Flow Cytometry
    • 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:

    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

    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)1

    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)2

    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)2

    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)

    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 18 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.

    Toggle Cloak
    Treatment

    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 ___%. Data is available

     L7ae Tuning

    GOAL: Test the sensitivity of L7ae repression

    21 complete Day One: Seed cells 28 complete Day Two: Transfect MAV1212-hEF1a-eBFP2, Wild Type hEF1a-2xKturn-eGFP, MAV1212-hEF1a-L7ae, pEXPR: hEF1a-rtTA (169.0 ng/uL), GG-Donor (dummy DNA) all at ~500 ng/uL into HEK cells 29 complete Day Three: DOX Induction 30 complete Day Four (___ am): Flow Cytometry
    • DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), Wild Type CAG-2xKturn-eGFP (0.96 uL), MAV1212-TRE-L7ae (0.89 uL), pEXPR: hEF1a-rtTA (0.27 uL), GG-Donor (to 1 ug)

    24-WELL PLATE TRANSFECTION MAP:

    1.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    Dummy DNA (1.75 fmole = 1.62 uL)

    2.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    Dummy DNA (1.75 fmole = 1.62 uL)

    3.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

    Dummy DNA (0.75 fmole = 0.70 uL)

    4.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

    Dummy DNA (0.75 fmole = 0.70 uL)

     

     

    //////

     

     

    //////

    5.

    pEXPR: hEF1a-rtTA

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

    MAV1212-TRE-L7ae (1.5 fmoles)

    0 uM DOX

    6.

    pEXPR: hEF1a-rtTA

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

    MAV1212-TRE-L7ae (1.5 fmoles)

    1X DOX

    7.

    pEXPR: hEF1a-rtTA

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

    MAV1212-TRE-L7ae (1.5 fmoles)

    10X DOX

    8.

    pEXPR: hEF1a-rtTA

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

    MAV1212-TRE-L7ae (1.5 fmoles)

    100X DOX

    9.

    pEXPR: hEF1a-rtTA

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

    MAV1212-TRE-L7ae (1.5 fmoles)

    1000X DOX

    10.

    pEXPR: hEF1a-rtTA

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

    MAV1212-TRE-L7ae (1.5 fmoles)

    2000X DOX

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

    11.

    Dummy DNA (1 ug = ___ uL)

    With Lipofectamine


    12.

    Dummy DNA (1 ug = ___ uL)

    With Lipofectamine


    13.

    No DNA

    No Lipofectamine

    14.

    No DNA

    No Lipofectamine

     

    //////

     

    //////


    RESULT:Transfection efficiency ___%. Data can be found .

     L7ae Quantification

    GOAL: Use TASBE tools to quantify the repression of L7ae

    32 complete 8/7/14: Seed cells 9 complete 8/8/14: Transfect hEF1a-rtTA (200.0 ng/uL), TRE-L7ae (200 ng/uL), Wild Type 2x-Kturn eGFP (200 ng/uL), TRE-mKate (200.0 ng/ uL), hEF1a-eBFP (200.0 ng/uL), hEF1a-mKate (200.0 ng/uL), hEF1a-eYFP (200.0 ng/uL), and GG Donor (200 ng/uL) into HEK cells 31 complete 8/10/14: (2 pm): Flow Cytometer

     

    • DNA Dilution: hEF1a-rtTA (0.60 uL), TRE-L7ae (1.48 uL), Wild Type 2x-Kturn eGFP (1.19 uL), TRE-mKate (1.0 uL), hEF1a-eBFP (0.93 uL), hEF1a-mKate (0.93 uL), hEF1a-eYFP (0.93 uL)

    24-WELL PLATE TRANSFECTION MAP:

    1. Neg Ctrl

    Dummy DNA

    With Lipofectamine


    2. Neg Ctrl

    Dummy DNA

    With Lipofectamine


    3. Neg/Neg Ctrl

    No DNA

    No Lipofectamine

    4. Neg/Neg Ctrl

    No DNA

    No Lipofectamine

    		  

    5.

    pEXPR: hEF1a-eYFP (1.0 fmoles)

    Dummy DNA (1.5 fmoles = 4.4 uL)

     

    6.

    pEXPR: hEF1a-mKate (1.0 fmoles)

    Dummy DNA (1.5 fmoles = 4.4 uL)

    7.

    pEXPR: hEF1a-eBFP (1.0 fmoles)

    Dummy DNA (1.5 fmoles = 4.4 uL)

    8.

    pEXPR: hEF1a-eYFP (1.0 fmoles)

    pEXPR: hEF1a-mKate (1.0 fmoles)

    pEXPR: hEF1a-eBFP (1.0 fmoles)

    Dummy DNA (0.75 fmoles = 2.2 uL)

    9.

    hEF1a-eBFP2 (0.5 fmole)

    Wild Type CAG-2xKturn-eGFP (1.0 fmoles)

    Dummy DNA (1.5 fmoles = 2.4 uL)

     

          

    10.

    pEXPR: hEF1a-rtTA (1.0 fmoles)

    hEF1a-eBFP2 (0.5 fmole)

    Wild Type CAG-2xKturn-eGFP (1.0 fmoles)

    MAV1212-TRE-L7ae (1.0 fmoles)

    pEXPR: TRE-mKate (1.0 fmoles)

    0 uM DOX

    11.

    pEXPR: hEF1a-rtTA (1.0 fmoles)

    hEF1a-eBFP2 (0.5 fmole)

    Wild Type CAG-2xKturn-eGFP (1.0 fmoles)

    MAV1212-TRE-L7ae (1.0 fmoles)

    pEXPR: TRE-mKate (1.0 fmoles)

    1X DOX

    12.

    pEXPR: hEF1a-rtTA 1.0 fmoles)

    hEF1a-eBFP2 (0.5 fmole)

    Wild Type CAG-2xKturn-eGFP (1.0 fmoles)

    MAV1212-TRE-L7ae (1.0 fmoles)

    pEXPR: TRE-mKate (1.0 fmoles)

    10X DOX

    13.

    pEXPR: hEF1a-rtTA (1.0 fmoles)

    hEF1a-eBFP2 (0.5 fmole)

    Wild Type CAG-2xKturn-eGFP (1.0 fmoles)

    MAV1212-TRE-L7ae (1.0 fmoles)

    pEXPR: TRE-mKate (1.0 fmoles)

    100X DOX

    14.

    pEXPR: hEF1a-rtTA (1.0 fmoles)

    hEF1a-eBFP2 (0.5 fmole)

    Wild Type CAG-2xKturn-eGFP (1.0 fmoles)

    MAV1212-TRE-L7ae (1.0 fmoles)

    pEXPR: TRE-mKate (1.0 fmoles)

    1000X DOX

    15.

    pEXPR: hEF1a-rtTA (1.0 fmoles)

    hEF1a-eBFP2 (0.5 fmole)

    Wild Type CAG-2xKturn-eGFP (1.0 fmoles)

    MAV1212-TRE-L7ae (1.0 fmoles)

    pEXPR: TRE-mKate (1.0 fmoles)

    2000X DOX


    RESULT: Transfection efficiency ___%. Data can be found

     L7ae Sensors (without siRNA)

    GOAL: Test the sensors in absence of siRNA

    10 complete 8.11.14: Seed cells 11 complete 8.12.14: Transfect 1 fmole of Low Sensor miR-144, Low Sensor miR-181c, High Sensor miR-30d, High Sensor miR-146a, High Sensor 125b, and High Sensor let-7f and 0.5 fmoles of pEXPR: hEF1a-mKate all at 200 ng/uL into HEK cells in addition to Dummy DNA (GG Donor) to fill the wells to 1 ug 12 complete 8.14.14: (4 pm): Flow Cytometer

     

    • DNA Dilution: hEF1a-mKate (0.46 uL), Wild Type Kturn-eGFP (1.19 uL), Low Sensor miR-144 (1.29 uL),Low Sensor miR-181c (1.29 uL), High Sensor miR-30d (1.37 uL), High Sensor miR-146a (1.37 uL), High Sensor 125b (1.37 uL), High Sensor let-7f (1.37 uL)


    24-WELL PLATE TRANSFECTION MAP:

     

    1. miR-144

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    Dummy DNA (2.9 uL = 1 fmoles)

    2. miR-181c

    pEXPR: hEF1a-mKate 

    Low Sensor miR-181c

    Dummy DNA (2.9 uL = 1 fmoles)

    3. miR-30d

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-30d

    Dummy DNA (2.2 uL = 0.75 fmoles)

    4. miR-146a

    pEXPR: hEF1a-mKate 

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-146a

    Dummy DNA (2.2 uL = 0.75 fmoles)

    5. miR-125b

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-125b

    Dummy DNA (2.2 uL = 0.75 fmoles)

    6. let-7f

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor let-7f

    Dummy DNA (2.2 uL = 0.75 fmoles)

    1. miR-144

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    Dummy DNA (2.9 uL = 1 fmoles)

    2. miR-181c

    pEXPR: hEF1a-mKate 

    Low Sensor miR-181c

    Dummy DNA (2.9 uL = 1 fmoles)

    3. miR-30d

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-30d

    Dummy DNA (2.2 uL = 0.75 fmoles)

    4. miR-146a

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-146a

    Dummy DNA (2.2 uL = 0.75 fmoles)

    5. miR-125b

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-125b

    Dummy DNA (2.2 uL = 0.75 fmoles)

    6. let-7f

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor let-7f (

    Dummy DNA (2.2 uL = 0.75 fmoles)

    //////

    //////

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

    13.

    pEXPR: hEF1a-mKate

    Dummy DNA (5.1uL = 1.75 fmoles)

    13.

    pEXPR: hEF1a-mKate

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

    Dummy DNA (3.6 uL = 1.25 fmoles)

    19.

    Dummy DNA

    With Lipofectamine

    20.

    Dummy DNA

    With Lipofectamine

    21.

    No DNA

    No Lipofectamine

    		22.

    No DNA

    No Lipofectamine


     

    RESULT: Transfection efficiency ___%. Data can be found .

     L7ae Sensors (with siRNA)

    GOAL: Test the sensors in the presence of siRNA

    -1 incomplete 33 complete 8.12.14: Seed cells 34 complete 8.13.14: Transfect 1 fmole of Low Sensor miR-144, Low Sensor miR-181c, High Sensor miR-30d, High Sensor miR-146a, High Sensor 125b, and High Sensor let-7f and 0.5 fmoles of pEXPR: hEF1a-mKate all at 200 ng/uL into HEK cells in addition to Dummy DNA (GG Donor) to fill the wells to 1 ug. 35 complete 8.15.14: (4 pm): Flow Cytometer
    • DNA Dilution: hEF1a-mKate (0.46 uL), Wild Type Kturn-eGFP (1.19 uL), Low Sensor miR-144 (1.29 uL),Low Sensor miR-181c (1.29 uL), High Sensor miR-30d (1.37 uL), High Sensor miR-146a (1.37 uL), High Sensor 125b (1.37 uL), High Sensor let-7f (1.37 uL), 5 uL Mature 144, 5 uL Mature 181c, 5 uL Mature 30d, 5 uL Mature 146a, 5 uL Mature 125b and 5 uL Mature let-7f


    24-WELL PLATE TRANSFECTION MAP:

    1. miR-144

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    siRNA-144

    Dummy DNA (2.9 uL = 1 fmoles)

    2. miR-181c

    pEXPR: hEF1a-mKate 

    Low Sensor miR-181c

    siRNA-181c

    Dummy DNA (2.9 uL = 1 fmoles)

    3. miR-30d

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-30d

    siRNA-30d

    Dummy DNA (2.2 uL = 0.75 fmoles)

    4. miR-146a

    pEXPR: hEF1a-mKate 

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-146a

    siRNA-146a

    Dummy DNA (2.2 uL = 0.75 fmoles)

    5. miR-125b

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-125b

    siRNA-125b

    Dummy DNA (2.2 uL = 0.75 fmoles)

    6. let-7f

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor let-7f

    siRNA-7f

    Dummy DNA (2.2 uL = 0.75 fmoles)

    1. miR-144

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    siRNA-144

    Dummy DNA (2.9 uL = 1 fmoles)

    2. miR-181c

    pEXPR: hEF1a-mKate 

    Low Sensor miR-181c

    siRNA-181c

    Dummy DNA (2.9 uL = 1 fmoles)

    3. miR-30d

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-30d

    siRNA-30d

    Dummy DNA (2.2 uL = 0.75 fmoles)

    4. miR-146a

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-146a

    siRNA-46a

    Dummy DNA (2.2 uL = 0.75 fmoles)

    5. miR-125b

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-125b

    siRNA-125b

    Dummy DNA (2.2 uL = 0.75 fmoles)

    6. let-7f

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor let-7f

    siRNA-7f

    Dummy DNA (2.2 uL = 0.75 fmoles)

    //////

    //////

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

    13.

    pEXPR: hEF1a-mKate

    Dummy DNA (5.1uL = 1.75 fmoles)

    13.

    pEXPR: hEF1a-mKate

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

    Dummy DNA (3.6 uL = 1.25 fmoles)

    19.

    Dummy DNA

    With Lipofectamine

    20.

    Dummy DNA

    With Lipofectamine

    21.

    No DNA

    No Lipofectamine

    		22.

    No DNA

    No Lipofectamine



    RESULT: Transfection efficiency ___%. Data can be found .

    L7ae Sensors (with and without siRNA)

    GOAL: Test the sensors in the presence and absence of siRNA

    -1 incomplete 14 incomplete Day 1: Seed cells 15 incomplete Day 2: Transfect 1 fmole of Low Sensor miR-144, Low Sensor miR-181c, High Sensor miR-30d, High Sensor miR-146a, High Sensor 125b, and High Sensor let-7f and 0.5 fmoles of pEXPR: hEF1a-mKate all at 200 ng/uL into HEK cells in addition to Dummy DNA (GG Donor) to fill the wells to 1 ug. 16 incomplete Day 4: (___ pm): Flow Cytometer
    • DNA Dilution: hEF1a-mKate (0.46 uL), Wild Type Kturn-eGFP (1.60 uL), Low Sensor miR-144 (1.29 uL),Low Sensor miR-181c (1.39 uL), High Sensor miR-30d (1.01 uL), High Sensor miR-146a (1.16 uL), High Sensor 125b (1.47 uL), High Sensor let-7f (1.37 uL), 5 uL Mature 144, 5 uL Mature 181c, 5 uL Mature 30d, 5 uL Mature 146a, 5 uL Mature 125b and 5 uL Mature let-7f


    24-WELL PLATE TRANSFECTION MAP:

    1. miR-144

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    Dummy DNA (2.9 uL = 1 fmoles)

    2. miR-181c

    pEXPR: hEF1a-mKate 

    Low Sensor miR-181c

    Dummy DNA (2.9 uL = 1 fmoles)

    3. miR-30d

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-30d

    Dummy DNA (2.2 uL = 0.75 fmoles)

    4. miR-146a

    pEXPR: hEF1a-mKate 

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-146a

    Dummy DNA (2.2 uL = 0.75 fmoles)

    5. miR-125b

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-125b

    Dummy DNA (2.2 uL = 0.75 fmoles)

    6. let-7f

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor let-7f

    Dummy DNA (2.2 uL = 0.75 fmoles)

    1. miR-144

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    siRNA-144

    Dummy DNA (2.9 uL = 1 fmoles)

    2. miR-181c

    pEXPR: hEF1a-mKate 

    Low Sensor miR-181c

    siRNA-181c

    Dummy DNA (2.9 uL = 1 fmoles)

    3. miR-30d

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-30d

    siRNA-30d

    Dummy DNA (2.2 uL = 0.75 fmoles)

    4. miR-146a

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-146a

    siRNA-146a

    Dummy DNA (2.2 uL = 0.75 fmoles)

    5. miR-125b

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor miR-125b

    siRNA-125b

    Dummy DNA (2.2 uL = 0.75 fmoles)

    6. let-7f

    pEXPR: hEF1a-mKate

    Wild Type CAG-2xKturn-eGFP

    High Sensor let-7f

    siRNA-7f

    Dummy DNA (2.2 uL = 0.75 fmoles)

    //////

    //////

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

    13.

    pEXPR: hEF1a-mKate

    Dummy DNA (5.1uL = 1.75 fmoles)

    13.

    pEXPR: hEF1a-mKate

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

    Dummy DNA (3.6 uL = 1.25 fmoles)

    19.

    Dummy DNA

    With Lipofectamine

    20.

    Dummy DNA

    With Lipofectamine

    21.

    No DNA

    No Lipofectamine

    		22.

    No DNA

    No Lipofectamine



    RESULT: Transfection efficiency ___%. Data can be found .


     L7ae Sensors (Multi Input)

    GOAL: Test the sensors in the presence and absence of siRNA

    -1 incomplete 20 complete 9.23.14: Seed cells 42 complete 9.24.14: Transfect 1 fmole of Low Sensor miR-144, High Sensor miR-30d, 0.5 fmoles pEXPR: hEF1a-mKate all at 200 ng/uL into HEK cells in addition to Dummy DNA (GG Donor) to fill the wells to 1 ug. 43 complete 9.26.14: (___ pm): Flow Cytometry
    • DNA Dilution: hEF1a-mKate (0.46 uL), Wild Type Kturn-eGFP (1.19 uL), Low Sensor miR-144 (1.29 uL),High Sensor miR-30d, 5 uL Mature 144, 5 uL Mature 30d


    24-WELL PLATE TRANSFECTION MAP:

     

     

    1.

    Untransfected

     

    2.

    pEXPR: hEF1a-mKate 

    Low Sensor miR-144c

    Dummy DNA (2.9 uL = 1 fmoles)

     

     

    //////



    //////



    //////



    //////

    3.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    Dummy DNA (2.9 uL = 1 fmoles)

    4.

    pEXPR: hEF1a-mKate 

    High Sensor miR-30d

    Dummy DNA (2.9 uL = 1 fmoles)5

    5.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    High Sensor miR-30d

    Wild Type CAG-2xKturn-eGFP

    6.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    High Sensor miR-30d

    Wild Type CAG-2xKturn-eGFP

    siRNA-30d

    7.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    High Sensor miR-30d

    Wild Type CAG-2xKturn-eGFP

    siRNA-144

    8.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    High Sensor miR-30d

    Wild Type CAG-2xKturn-eGFP

    siRNA-30d

    siRNA-144

    //////

    9.

    pEXPR: hEF1a-mKate 

    High Sensor miR-125b

    Dummy DNA (2.9 uL = 1 fmoles)5

    10.

    pEXPR: hEF1a-mKate

    Low Sensor miR-125b

    High Sensor miR-30d

    Wild Type CAG-2xKturn-eGFP

    11.

    pEXPR: hEF1a-mKate

    Low Sensor miR-125b

    High Sensor miR-30d

    Wild Type CAG-2xKturn-eGFP

    siRNA-30d

    12.

    pEXPR: hEF1a-mKate

    Low Sensor miR-125b

    High Sensor miR-30d

    Wild Type CAG-2xKturn-eGFP

    siRNA-125b

    13.

    pEXPR: hEF1a-mKate

    Low Sensor miR-125b

    High Sensor miR-30d

    Wild Type CAG-2xKturn-eGFP

    siRNA-30d

    siRNA-125b

    //////

    14.

    pEXPR: hEF1a-mKate 

    High Sensor miR-181c

    Dummy DNA (2.9 uL = 1 fmoles)5

    15.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    High Sensor miR-181c

    Wild Type CAG-2xKturn-eGFP

    16.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    High Sensor miR-181c

    Wild Type CAG-2xKturn-eGFP

    siRNA-181c

    17.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    High Sensor miR-181c

    Wild Type CAG-2xKturn-eGFP

    siRNA-144

    18.

    pEXPR: hEF1a-mKate

    Low Sensor miR-144

    High Sensor miR-181c

    Wild Type CAG-2xKturn-eGFP

    siRNA-181c

    siRNA-144

          


    RESULT: Transfection efficiency ___%. Data can be found .

    TAL14 Repression

    GOAL: Test the strength of TAL14

    44 complete 7/25/14: Seed cells 45 complete 7/26/14: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB (dummy DNA) at ~500 ng/uL into HEK cells 46 complete 7/28/14 (4 pm): Flow Cytometry

     

    • DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), TAL14-mKate (0.62 uL), hEF1a-GAL4VP16 (0.72 uL), hEF1a-TAL14 (___ uL), Dummy DNA (to 1 ug)


    24-WELL PLATE TRANSFECTION MAP:

    1. (B)1

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    Dummy DNA (2.5 fmoles = 0.76 uL)

    2. (B)2

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    Dummy DNA (2.5 fmoles = 0.76 uL)

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

    //////

     

    3. Neg Ctrl

    Dummy DNA (3.2 fmoles = 2.05 uL)

    With Lipofectamine

    4. (B + R)1

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    Dummy DNA (2.0 fmoles = 0.61 uL)

    5. (B + R)2

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    Dummy DNA (2.0 fmoles = 0.61 uL)

    //////

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

    6. Neg Ctrl

    Dummy DNA (3.2 fmoles = 2.05 uL)

    With Lipofectamine

    7. (B + R + GAL)1

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.5 fmoles)

    8. (B + R + GAL)2

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.5 fmoles)

    //////

    //////

     

    		//////

    9. Neg/Neg Ctrl

    No DNA

    No Lipofectamine

    10. (B + R + GAL)1

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.5 fmoles)

    hEF1a-TAL14 (1.5 fmoles)

    11. (B + R + GAL)2

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.5 fmoles)

    hEF1a-TAL14 (1.5 fmoles)

    //////

    		////////////12. Neg/Neg Ctrl

    No DNA

    No Lipofectamine

     

    Results: Transfection efficiency ___%. Data can be found

     TAL14 Tuning

    GOAL: Determine how much TAL14 is needed to repress TAL14-UAS-mKate

    26 complete Day One: Seed cells 27 complete Day Two: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB (dummy DNA) at ~500 ng/uL into HEK cells 25 complete Day Four (12:30 pm): Flow Cytometry

     

    • DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), TAL14-mKate (0.62 uL), hEF1a-GAL4VP16 (0.72 uL), hEF1a-TAL14 (0.22 uL = 1 fmol), Dummy DNA (to 1 ug)


    24-WELL PLATE TRANSFECTION MAP:

    1. 

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    Dummy DNA (1.75 fmoles = 4.06 uL)

    2. 

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    Dummy DNA (1.75 fmoles = 04.06 uL)

    3.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    Dummy DNA (1.0 fmoles = 2.32 uL)

    4.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    Dummy DNA (1.0 fmoles = 2.32 uL)

    5.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.5 fmoles)

    Dummy DNA (0.75 fmoles = 1.74 uL)

    6.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.5 fmoles)

    Dummy DNA (0.75 fmoles = 1.74 uL)

    7.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (1.0 fmoles)

    8.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (1.5 fmoles)

    9.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (2.0 fmoles)

    10.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (2.5 fmoles)

    11.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (3.0 fmoles)

    12.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (3.5 fmoles)

    13.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (4.0 fmoles)

    14.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (4.5 fmoles)

    15.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (5.0 fmoles)

    16.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (10.0 fmoles)

    17.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (15.0 fmoles)

    18.

    MAV1212-hEF1a-eBFP2 (1.0 fmole)

    UAS-TAL14-mKate (1.5 fmoles)

    hEF1a-Gal4VP16 (1.0 fmoles)

    hEF1a-TAL14 (20.0 fmoles)

    19.

    Dummy DNA (3.2 fmoles = 2.05 uL)

    With Lipofectamine

    20.

    Dummy DNA (3.2 fmoles = 2.05 uL)

    With Lipofectamine

    21.

    No DNA

    No Lipofectamine

    		22.

    No DNA

    No Lipofectamine

    		  

    RESULT: Transfection efficiency ___%. Data can be found here.

    Toggle Cloak
    Treatment

    cloak
    Cloak

    Testing effectiveness of miRNA1–miRNA4 in down-regulating BACE1 expression v1

    A1

      • Hef1a: eBFP 250ng
      • Dummy DNA 750ng


        Positive Control

    A2

      • Hef1a: eBFP 250ng
      • Hef1a: BACE1 250ng
      • Dummy DNA 500ng

    Negative Control

    A3

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        0 nM Dox

    A4

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A5

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        1000 nM Dox

    A6

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    A7

    • Hef1a:mKATE 250ng
    • Hef1a:BFP 250ng
    • Dummy DNA 500ng
    		     
          
          

    Testing inducible expression of BACE2

    B1

    • Hef1a:eBFP 333ng
    • Dummy DNA 666ng

    Negative Control

    B2

    • Hef1a:eBFP 333ng
    • Hef1a:rtTA 333ng
    • TRE:BACE2 333ng

    0 nM Dox

    B3

    • Hef1a:eBFP 333ng
    • Hef1a:rtTA 333ng
    • TRE:BACE2 333ng

    1000 nM Dox

    B4

    • No DNA (untransfected)

     

    Double negative control

    		  
          
          
          

    Transfection: Week of 8/11

    A1

      • Hef1a: eBFP 250ng
      • Dummy DNA 750ng



    Negative BACE1 Control

    A2

      • Hef1a: eBFP 250ng
      • Hef1a: BACE1 250ng
      • Dummy DNA 500ng

     

    Positive BACE1 Control

     A3

      • Hef1a:mKATE 250ng
      • Hef1a:BFP 250ng
      • Dummy DNA 500ng

     

    mKATE control

    A4

      • Hef1:miRNAg1 250ng
      • Hef1a:BFP 250ng
      • Dummy DNA 500ng

     

    miRNA1g control

    A5

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        0 nM Dox

    A6

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A7

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        1000 nM Dox

    A8

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    A9

      • Hef1a: eBFP 250ng
      • TRE: miRNA2-mKATE250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        0 nM Dox

    A10

      • Hef1a: eBFP 250ng
      • TRE: miRNA2-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A11

      • Hef1a: eBFP 250ng
      • TRE: miRNA2-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        1000 nM Dox

    A12

      • Hef1a: eBFP 250ng
      • TRE: miRNA2-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    A13

      • Hef1a: eBFP 250ng
      • TRE: miRNA3-mKATE250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        0 nM Dox

    A14

      • Hef1a: eBFP 250ng
      • TRE: miRNA3-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A15

      • Hef1a: eBFP 250ng
      • TRE: miRNA3-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        1000 nM Dox

    A16

      • Hef1a: eBFP 250ng
      • TRE: miRNA3-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    A17

      • Hef1a: eBFP 250ng
      • TRE: miRNA4-mKATE250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        0 nM Dox

    A18

      • Hef1a: eBFP 250ng
      • TRE: miRNA4-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A19

      • Hef1a: eBFP 250ng
      • TRE: miRNA4-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Dummy DNA 250ng

        1000 nM Dox

    A20

      • Hef1a: eBFP 250ng
      • TRE: miRNA4-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    B1

      • Hef1a:eBFP 333ng
      • Dummy DNA 666ng

     

    Negative Control

    B2

      • Hef1a:eBFP 333ng
      • Hef1a:rtTA 333ng
      • TRE:BACE2 333ng

     

    0 nM Dox

    B3

      • Hef1a:eBFP 333ng
      • Hef1a:rtTA 333ng
      • TRE:BACE2 333ng

     

    1000 nM Dox

    B4

      • No DNA (untransfected)

     

     

    Double negative control

     Transfection 9/17

     

    A1

      • Hef1a: eBFP 250ng
      • Dummy DNA 750ng



    Negative BACE1 Control

    A2

      • Hef1a: eBFP 250ng
      • Hef1a: BACE1 250ng
      • Dummy DNA 500ng

     

    Positive BACE1 Control

     A3

      • Hef1a:mKATE 250ng
      • Hef1a:BFP 250ng
      • Dummy DNA 500ng

     

    mKATE control

    A4

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

     

    A5

      • Hef1a: eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    		 A6
      • Hef1a: eBFP 250ng
      • TRE: miRNA2-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A7

      • Hef1a: eBFP 250ng
      • TRE: miRNA2-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    A8

      • Hef1a: eBFP 250ng
      • TRE: miRNA3-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

     A9

      • Hef1a: eBFP 250ng
      • TRE: miRNA3-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    A10

      • Hef1a: eBFP 250ng
      • TRE: miRNA4-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A11

      • Hef1a: eBFP 250ng
      • TRE: miRNA4-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    B1

      • Hef1a:eBFP 333ng
      • Dummy DNA 666ng

     

    Negative Control

    B2

      • Hef1a:eBFP 333ng
      • Hef1a:rtTA 333ng
      • TRE:BACE2 333ng

     

    0 nM Dox

    B3

      • Hef1a:eBFP 333ng
      • Hef1a:rtTA 333ng
      • TRE:BACE2 333ng

     

    1000 nM Dox

    		 B4
      • No DNA (untransfected)

     

    Double negative control