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L7ae 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


Attempt #2

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

 

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

24-WELL PLATE TRANSFECTION MAP:

(B)1

  • MAV1212-hEF1a-eBFP2

(B)2

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

(GM)1

  • Mutant Hef1a-2xKturn-eGFP

(GM)2

  • Mutant Hef1a-2xKturn-eGFP

(GM + B)1

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

(GM + B)2

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

(GM + B + L)1

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

(GM + B + L)2

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

(GWT)1

  • Wild Type Hef1a-2xKturn-eGFP

(GWT)2

  • Wild Type Hef1a-2xKturn-eGFP


(GWT + B)1

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

(GWT + B)2

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

(GWT + B + L)1

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

(GWT + B + L)2

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

Neg Ctrl

  • Dummy DNA
  • With Lipofectamine


Neg Ctrl

  • Dummy DNA
  • With Lipofectamine


Neg/Neg Ctrl

  • No DNA
  • No Lipofectamin

Neg/Neg Ctrl

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

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


Attempt #3

17 complete 7/24/14: 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)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)

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

11. Neg Ctrl

hEF1a-PirB (3.2 fmoles = 2.05 uL)

With Lipofectamine


12. Neg Ctrl

hEF1a-PirB (3.2 fmoles = 2.05 uL)

With Lipofectamine


13. Neg/Neg Ctrl

No DNA

No Lipofectamine

14. Neg/Neg Ctrl

No DNA

No Lipofectamine

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

RESULT: Transfection efficiency ___%. FACS data is available

TAL14 Repression

GOAL: Test the strength of TAL14

14 complete 7/25/14: Seed cells 15 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 16 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

 Tuning L7ae Repression

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

 Tuning TAL14 Repression

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

L7ae Sensors (without siRNA)

GOAL: Test the sensors in absence of siRNA

10 complete 7/26/14: Seed cells 11 incomplete 7/27/14: 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) and High Sensor let-7f (889.6 ng/uL) into HEK cells 12 incomplete 7/29/14: (4 pm): Flow Cytometer

 

  • DNA Dilution: 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)


24-WELL PLATE TRANSFECTION MAP:

1. miR-144

Low Sensor miR-144 ()

2. miR-181c

Low Sensor miR-181c (1.5 fmoles)

3. miR-30d

High Sensor miR-30d (1.5 fmoles)

4. miR-146a

High Sensor miR-146a (1.5 fmoles)

5. miR-125b

High Sensor miR-125b (1.5 fmoles)

6. let-7f

High Sensor let-7f (1.5 fmoles)

7. miR-144

Low Sensor miR-144 (1.5 fmoles)

8. miR-181c

Low Sensor miR-181c (1.5 fmoles)

9. miR-30d

High Sensor miR-30d (1.5 fmoles)

10. miR-146a

High Sensor miR-146a (1.5 fmoles)

11. miR-125b

High Sensor miR-125b (1.5 fmoles)

12. let-7f

High Sensor let-7f (1.5 fmoles)

//////

//////

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

Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine


Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

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.

L7ae Sensors (with siRNA)

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:

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.

Quantifying L7ae Repression

GOAL: Quantify the repression of L7ae

34 incomplete complete Day 1: Seed cells 35 incomplete Day 2: Transfect hEF1a-rtTA (169200.0 ng/uL), TRE-L7ae (500 200 ng/uL), Wild Type 2x-Kturn eGFP (500 200 ng/uL), TRE-mKate (431200.0 ng/ uL), hEF1a-eBFP (405200.0 ng/uL), hEF1a-mKate (621200.0 ng/uL), hEF1a-eYFP (1044.8 200.0 ng/uL), and GG Donor (200 ng/uL) into HEK cells 31 incomplete Day 4: (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:

151. Neg Ctrl

Dummy DNA

With Lipofectamine


162. Neg Ctrl

Dummy DNA

With Lipofectamine


173. Neg/Neg Ctrl

No DNA

No Lipofectamine

184. Neg/Neg Ctrl

No DNA

No Lipofectamine

  

115.

pEXPR: hEF1a-eYFP (1.0 fmoles)

Dummy DNA (1.5 fmoles = 4.4 uL)

 

126.

pEXPR: hEF1a-mKate (1.0 fmolesfmoles)

Dummy DNA (1.5 fmoles = 4.4 uL)

137.

pEXPR: hEF1a-eBFP (1.0 fmoles)

Dummy DNA (1.5 fmoles = 4.4 uL)

148.

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)

39.

hEF1a-eBFP2 (0.5 fmole)

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

Dummy DNA (___ fmole = ___ 1.5 fmoles = 2.4 uL)

 

      

510.

pEXPR: hEF1a-rtTA

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

611.

pEXPR: hEF1a-rtTA

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

712.

pEXPR: hEF1a-rtTA

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

813.

pEXPR: hEF1a-rtTA

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

914.

pEXPR: hEF1a-rtTA

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

1015.

pEXPR: hEF1a-rtTA

hEF1a-eBFP2 (05 fmole)

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

MAV1212-TRE-L7ae (1.0 fmoles)

pEXPR: TRE-mKate (1.0 fmoles)

2000X DOX

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

Toggle Cloak
Treatment

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

  
      
      
      

 


 

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