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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: 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 0 complete 7/1917/14 (10 am12 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-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 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 + 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-eGFPMAV1212
  • Wild Type Hef1a-hEF1a2xKturn-eBFP2eGFP
  • MAV1212-hEF1a-L7aeeBFP2

(GM + B + L)2

  • Mutant Hef1a-2xKturn-eGFPMAV1212
  • Wild Type Hef1a-hEF1a2xKturn-eBFP2eGFP
  • MAV1212-hEFahEF1a-L7aeeBFP2

(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
  • MAV1212Mutant Hef1a-hEF1a2xKturn-eBFP2eGFP
  • MAV1212-hEFa1hEF1a-L7aeeBFP2

(GWT + B + L)2

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212Mutant Hef1a-hEF1a2xKturn-eBFP2eGFP
  • MAV1212-hEF1a-L7aeeBFP2

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

Inconclusive. No L7ae was added.


Attempt #2

2 complete 7/16 17 complete 7/21/14: Seed cells 18 incomplete 1 complete 7/2217/14: 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 complete 7/2319/14 (10 am): Flow Cytometer

 

  • DNA Dilution: 5 uL MAV1212-hEF1a-eBFP2 (0.11 uL), Wild Type CAG, 10 uL Mutant Hef1a-2xKturn-eGFP (1.12 uL), Mutant CAG, 20 uL Wild Type Hef1a-2xKturn-eGFP (0.58 uL), , 3 uL MAV1212-hEF1a-L7ae (0.14 uL), hEF1a-PirB (dummy DNA to 1 ug)

24-WELL PLATE TRANSFECTION MAP:

1. 3. BWT + 2MAV1212hEF1aWT (0.5 fmole)Wild Type CAG(1 fmole)PirB (0.99 uL)5. WT

MAV1212-hEF1a-eBFP2 (0.5 fmole)

Wild Type CAG(1 fmole)L7ae (0.14 uL)

hEF1a-PirB (0.57 uL)

6. (GWT (0.5 fmole) CAG7. (GM (0.5 fmole)Multant CAG(1 fmole)PirB (0.99 uL)8. M (0.5 fmole)

Multant CAG-2xKturn-eGFP (1 fmole)

hEF1a-PirB (0.99 uL)

9. (GM 1(0.5 fmole)Multant CAG-2xKturn-eGFP (1 fmole)(1 fmole)

hEF1a-PirB (0.57 uL)

11. Neg Ctrl

hEF1a-PirB (1.76 uL)

With Lipofectamine

12. Neg Ctrl

hEF1a-PirB (1.76 uL)

With Lipofectamine

(B)1

  • MAV1212-hEF1a-eBFP2

(0.5 fmoleB)2

hEF1a-PirB (1.55 uL)

2. (B)2

MAV1212-hEF1a-eBFP2 (0.5 fmole)

hEF1a-PirB (1.55 uL)

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

(G

M)

1

  • Mutant Hef1a-
  • 2xKturn-eGFP

(GM)2

  • Mutant Hef1a
eBFP2 (0.5 fmole)Wild Type CAG
  • -2xKturn-eGFP
(1 fmole)hEF1a-PirB (0.99 uL)
4.

(G

M + B)1

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

(GM + B)2

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

(G

M + 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

(1 fmoleGWT)2MAV1212

  • Wild Type Hef1a-
hEF1a-L7ae (1 fmole)

hEF1a-PirB (0.57 uL)

////////////
  • 2xKturn-eGFP


(GWT

+ B)1

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

(GWT + B)2

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

(G

WT + 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

10. (GM + B + L)2

MAV1212-hEF1a-eBFP2 (0.5 fmole)

Multant CAG-2xKturn-eGFP (1 fmole)

MAV1212-hEF1a-L7ae (1 fmole)

hEF1a-PirB (0.57 uL)

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

13. Neg/Neg Ctrl

No DNA

No 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

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: 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, 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)

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:

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)

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

 

Neg Ctrl

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

3. (GWT + B

(B + Y

)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

  • UAS-TAL14-eYFP
    • (B + Y

    )2

    MAV1212-hEF1a-eBFP2

  • UAS-TAL14-eYFP

    • (B + R)1

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

    (1.0 fmole)

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

    hEF1a-PirB (1.0 fmole = 0.99 uL)

    5. (GWT + B + L)1

    (B + R)2

    MAV1212-hEF1a-eBFP2

  • UAS-TAL14-mKate
    • //////

    Neg Ctrl

      • Dummy DNA
      • With Lipofectamine

    (1.0 fmole)

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

    MAV1212-hEF1a-L7ae (1.5 fmoles)

    6. (GWT + B + L)2

    (B + Y + GAL)1

    MAV1212-hEF1a-eBFP2

  • UAS-TAL14-eYFP
  • hEF1a-Gal4VP16

    • (1.0 fmole)

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

    (B + Y + GAL)2

    MAV1212-hEF1a-

    eBFP2

    L7ae (1.5 fmoles)

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

    7. (GM + B

  • UAS-TAL14-eYFP
  • hEF1a-Gal4VP16
    • (B + R + GAL

    )1

    MAV1212-hEF1a-eBFP2

    (1.0 fmole)

    Multant CAG-2xKturn-eGFP (1.5 fmoles)

    UAS-TAL14-mKate

    hEF1a-

    Gal4VP16

    PirB (1.0 fmole = 0.99 uL)

    8. (GM + B

    (B + R + GAL

    )2

    MAV1212-hEF1a-eBFP2

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

    Neg/Neg Ctrl

      • No DNA
      • No Lipofectamin

    (1.0 fmole)

    Multant CAG-2xKturn-eGFP (1.5 fmoles)

    hEF1a-PirB (1.0 fmole = 0.99 uL)

    9. (GM + B + L

    (B + Y + GAL

    )1

    MAV1212-hEF1a-eBFP2

  • UAS-TAL14-eYFP
  • hEF1a-Gal4VP16
  • hEF1a-TAL14

    • (1.0 fmole)

    Multant CAG-2xKturn-eGFP (1.5 fmoles)

    MAV1212-hEF1a-L7ae (1.5 fmoles)

    10. (GM + B + L

    (B + Y + GAL

    )2

    MAV1212-hEF1a-eBFP2

  • UAS-TAL14-eYFP
  • hEF1a-Gal4VP16
  • hEF1a-TAL14

    • (1.0 fmole)

    Multant CAG-2xKturn-eGFP (1.5 fmoles)

    (B + R + GAL)1

    MAV1212-hEF1a-

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

    • 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.

    (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.

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


    RESULT: Transfection efficiency ___%. Data is available

     L7ae Tuning Experiment #3

    GOAL: Test the sensors in absence of siRNAsensitivity of L7ae repression

    21 complete Day One 10 incomplete Day 1: Seed cells 11 incomplete 28 complete Day 2Two: Transfect Low Sensor miR-144 (___ ng/uL), Low Sensor miR-181c (___ ng/uL), High Sensor miR-30d (___ ng/uL), High Sensor miR-146a (___ MAV1212-hEF1a-eBFP2, Wild Type hEF1a-2xKturn-eGFP, MAV1212-hEF1a-L7ae, pEXPR: hEF1a-rtTA (169.0 ng/uL), High Sensor 125b (___ ng/uL) and High Sensor let-7f (___ GG-Donor (dummy DNA) all at ~500 ng/uL ) into HEK cells 12 incomplete 29 complete Day 4: (2 pmThree: DOX Induction 30 complete Day Four (___ am): Flow CytometerCytometry
       
      • 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:

      • 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:

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

      Neg Ctrl

        • Dummy DNA
        • With Lipofectamine

      Neg Ctrl

        • Dummy DNA
        • With Lipofectamine

      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)

       

       

      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/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.

      Toggle Cloak
      Treatment

      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