Versions Compared

Old Version 96

changes.mady.by.user Unknown User (gary_9@mit.edu)

Saved on

compared with

New Version Current

changes.mady.by.user Unknown User (gary_9@mit.edu)

Saved on

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.
Comment: Migration of unmigrated content due to installation of a new plugin

...

Toggle Cloak
miRNA Detection

 

 

Cloak

Experiment #1L7ae Repression

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 Inconclusive. No L7ae was added.


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 : 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)

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)21

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)12

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)12

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 Data is available

 

Experiment #2L7ae Tuning

GOAL: Test the strength sensitivity of TAL14L7ae repression

14 complete 7/25/1421 complete Day One: Seed cells 15 complete 7/26/14 28 complete Day Two: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB 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 16 complete 7/28/14 (4 pm 29 complete Day Three: DOX Induction 30 complete Day Four (___ am): Flow Cytometry 
  • DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), TAL14-mKate Wild Type CAG-2xKturn-eGFP (0.62 96 uL), hEF1aMAV1212-TRE-GAL4VP16 L7ae (0.72 89 uL), pEXPR: hEF1a-TAL14 (___ rtTA (0.27 uL), Dummy DNA GG-Donor (to 1 ug)

24-WELL PLATE TRANSFECTION MAP:

 (B)125 fmoles 076 (B)225 fmoles 076

//////

  Neg Ctrl

4. (B + R)1

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

UASTAL14mKate 20 fmoles 61 5. (B + R)2UASTAL14mKate 20 fmoles 61 //////6

7. (B + R + GAL)1

Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

UASTAL14mKate hEF1aGal4VP16

8. (B + R + GAL)2

MAV1212-hEF1a-UASTAL14mKate hEF1aGal4VP16

10. (B + R + GAL)1

UASTAL14mKate hEF1aGal4VP16 hEF1a-TAL14 11. (B + R + GAL)2UASTAL14mKate hEF1aGal4VP16 TAL14 5 fmoles)/12. Neg/Neg Ctrl

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)

//////

//////

 

//////

9. Neg/Neg Ctrl

No DNA

No Lipofectamine

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

 Experiment #3

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/

GOAL: Test the sensors in absence of siRNA

10 incomplete 7/26/14: Seed cells 11 incomplete 7/279 complete 8/8/14: Transfect Low Sensor miRhEF1a-144 rtTA (708200.2 0 ng/uL), Low Sensor miRTRE-181c (628.1 L7ae (200 ng/uL), Wild Type 2x-Kturn eGFP (200 ng/uL), TRE-mKate (200.0 ng/ uL), High Sensor miRhEF1a-30d eBFP (1046200.2 0 ng/uL), High Sensor miRhEF1a-146a mKate (828200.8 0 ng/uL), High Sensor 125b (1159.5 hEF1a-eYFP (200.0 ng/uL) and High Sensor let-7f (889.6 , and GG Donor (200 ng/uL) into HEK cells 12 incomplete 7/2931 complete 8/10/14: (4 2 pm): Flow Cytometer

 

  • DNA Dilution: Low Sensor miRhEF1a-144 (___ rtTA (0.60 uL), Low Sensor miR-181c (___ uL), High Sensor miR-30d (___ uL), High Sensor miR-146a (___ uL), High Sensor 125b (___ uL), High Sensor let-7f (___ 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:

miR-144

Low Sensor miR-144 ()

3. miR-30d

High Sensor miR-30d

4. miR-146a

High Sensor miR-146a 5

7. miR-144

Low Sensor miR-144 miR-181cLow Sensor miR-181c 5

9. miR-30d

High Sensor miR-30d 5

12. let-7f

High Sensor let-7f

Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

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

2. miR-181c

Low Sensor miR-181c (1.5 fmoles)

(1.5 fmoles = 4.4 uL)

 

6.

pEXPR: hEF1a-mKate

(1.

0 fmoles)

5. miR-125b

High Sensor miR-125b (1.5 fmoles)

6. let-7f

High Sensor let-7f (1.5 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)

10. miR-146a

High Sensor miR-146a (1.5 fmoles)

11. miR-125b

High Sensor miR-125b (1.5 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)

//////

//////

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

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

 

      

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)

GOAL: Test the sensors in the presence of siRNA

13 incomplete Day 1: Seed cells 9 incomplete Day 2: Transfect Low Sensor miR-144 (708.2 ng/uL), Low Sensor miR-181c (628.1 ng/uL), High Sensor miR-30d (1046.2 ng/uL), High Sensor miR-146a (828.8 ng/uL), High Sensor 125b (1159.5 ng/uL), High Sensor let-7f (889.6 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:

 

  • Mature 144
    •  
  • Mature 181c

    •  

    High Sensor
  • Mature 30d
    •  146a
  • Mature 146a
    •  30d
  • Mature 30d

    • Neg Ctrl

      • Dummy DNA
      • With Lipofectamine

    Neg Ctrl

      • Dummy DNA
      • With Lipofectamine

    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
      • Mature 125b

     

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

     

      • Low Sensor miR-144
      • Mature 144

     

      • Low Sensor miR-181c
      • Mature 181c

    High Sensor miR-

     

      • High Sensor miR-146a
      • Mature 146a

     

      • High Sensor miR-125b
      • Mature 125b

     

      • High Sensor let-7f
      • Mature 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 #5

    GOAL: Test the sensitivity of L7ae repression

    21 incomplete Day One: Seed cells 21 incomplete Day Two: 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 21 incomplete Day Three: DOX Induction 21 incomplete Day Four (___ 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:

          
          
          
          

    Toggle Cloak
    Treatment

    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

  • Hef1a: eBFP 250ng
    • TRE: miRNA1
    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

     

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

    Repeat for miRNA2, miRNA3, and miRNA4 (same arrangement of wells as shown here, just substituting out "miRNA1" for the appropriate miRNA)

    Hef1a: eBFP 1000ng
    Negative Control
      • Hef1a: eBFP 500ng
      • Hef1a: BACE1 500ng

    Positive Control

     

    		   
      • Hef1a: eBFP 333ng
      • TRE: miRNA1 333ng
      • Hef1a: rtTA 333ng

    0 nM Dox

    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

  • Hef1a: eBFP 333ng
    • TRE: miRNA1 333ng

     

    A5

      • Hef1a:
    rtTA 333ng

    1 nM Dox

    Hef1a:
      • eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng
    1

    1000 nM Dox

    		 A6
      • Hef1a: eBFP
    333ng
      • 250ng
      • TRE:
    miRNA1 333ng
      • miRNA2-mKATE 250ng
      • Hef1a: rtTA
    333ng10
      • 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A7

      • Hef1a: eBFP 250ng
      • TRE:
    miRNA1
      • miRNA2-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng
    10

    1000 nM Dox

    A8

      • Hef1a: eBFP
    333ng
      • 250ng
      • TRE:
    miRNA1 333ng
      • miRNA3-mKATE 250ng
      • Hef1a: rtTA
    333ng100
      • 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

     A9

      • Hef1a: eBFP 250ng
      • TRE:
    miRNA1
      • miRNA3-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng
    100

    1000 nM Dox

    A10

      • Hef1a: eBFP
    333ng
      • 250ng
      • TRE: miRNA4-mKATE 250ng
      • Hef1a: rtTA 250ng
    TRE: miRNA1 333ng
      • Hef1a:
    rtTA 333ng
      • BACE1 250ng
    1000

    0 nM Dox

    A11

      • Hef1a: eBFP 250ng
      • TRE:
    miRNA1 TRE: miRNA1
      • 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
    2000 TRE: miRNA1 250ng

     

    0 nM Dox

    B3

      • Hef1a:eBFP
    250ng
      • 333ng
      • Hef1a:rtTA
    250ng
      • 333ng
  • Hef1a: BACE1 250ng
    • 2000 nM Dox
      • TRE:BACE2 333ng

     

    1000 nM Dox

    		 B4
      • No DNA (untransfected)

     

    Double negative control