Page History

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Cloak

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

//////

(G_M)1

Mutant Hef1a-2xKturn-eGFP

(G_M)₂

Mutant Hef1a-2xKturn-eGFP

(G_M+ B)₁

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

(G_M+ B)₂

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

(G_M+ B + L)₁

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

(G_M+ B + L)₂

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

(G_WT)1

Wild Type Hef1a-2xKturn-eGFP

(G_WT)₂

Wild Type Hef1a-2xKturn-eGFP

(G_WT+ B)₁

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

(G_WT+ B)₂

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

(G_WT+ B + L)₁

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

(G_WT+ 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

//////

(G_M)1

Mutant Hef1a-2xKturn-eGFP

(G_M)₂

Mutant Hef1a-2xKturn-eGFP

(G_M+ B)₁

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

(G_M+ B)₂

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

(G_M+ B + L)₁

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

(G_M+ B + L)₂

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

(G_WT)1

Wild Type Hef1a-2xKturn-eGFP

(G_WT)₂

Wild Type Hef1a-2xKturn-eGFP

(G_WT+ B)₁

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

(G_WT+ 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

(G_WT+ 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. (G_WT+ 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. (G_WT+ 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. (G_WT+ B + L)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

MAV1212-hEF1a-L7ae (1.5 fmoles)

6. (G_WT+ B + L)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

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

MAV1212-hEF1a-L7ae (1.5 fmoles)

//////

7. (G_M+ B)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

8. (G_M+ B)2

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

hEF1a-PirB (1.0 fmole = 0.99 uL)

9. (G_M+ B + L)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Multant CAG-2xKturn-eGFP (1.5 fmoles)

MAV1212-hEF1a-L7ae (1.5 fmoles)

10. (G_M+ 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 1 fmole of 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 and 0.5 fmoles of pEXPR: hEF1a-mKate all at 200 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

pEXPR: hEF1a-mKate (1.0 fmoles)

Low Sensor miR-144 (1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

2. miR-181c

pEXPR: hEF1a-mKate (1.0 fmoles)

Low Sensor miR-181c (1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

3. miR-30d

pEXPR: hEF1a-mKate (1.0 fmoles)

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

High Sensor miR-30d(1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

4. miR-146a

pEXPR: hEF1a-mKate (1.0 fmoles)mKate

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

High Sensor miR-146a (1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

5. miR-125b

pEXPR: hEF1a-mKate(1.0 fmoles)

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

High Sensor miR-125b (1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

6. let-7f

pEXPR: hEF1a-mKate (1.0 fmoles)

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

High Sensor let-7f

Dummy DNA (___ uL = ___ fmoles)

1. miR-144

pEXPR: hEF1a-mKate

Low Sensor miR-144 1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

2. miR-181c

pEXPR: hEF1a-mKate (1.0 fmoles)

Low Sensor miR-181c(1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

3. miR-30d

pEXPR: hEF1a-mKate (1.0 fmoles)

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

High Sensor miR-30d (1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

4. miR-146a

pEXPR: hEF1a-mKate (1.0 fmoles)mKate

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

High Sensor miR-146a (1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

5. miR-125b

pEXPR: hEF1a-mKate(1.0 fmoles)

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

High Sensor miR-125b (1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

6. let-7f

pEXPR: hEF1a-mKate (1.0 fmoles)

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

High Sensor let-7f (1.0 fmoles)

Dummy DNA (___ uL = ___ fmoles)

//////

13.

pEXPR: hEF1a-mKate

Dummy DNA (___ uL = ___ fmoles)

13.

pEXPR: hEF1a-mKate

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

Dummy DNA (___ uL = ___ 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 (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 complete Day 1: Seed cells 35 incomplete Day 2: 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 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:
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: Results can be found here

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