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Toggle Cloak

miRNA Detection

RESULT: Results can be found here

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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

//////

(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 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

//////

(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 : 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. (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 Data is available

TAL14 Repression L7ae Tuning

GOAL: Test the strength sensitivity of TAL14L7ae repression

14

complete

7/25/14

21

complete

Day One: Seed cells

15

complete

7/26/14

28

complete

Day Two: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, Wild Type hEF1a-2xKturn-eGFP, MAV1212-hEF1a-GAL4VP16L7ae, pEXPR: hEF1a-TAL14, and hEF1a-PirB 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

4. (B + R)₁

MAV1212-hEF1a-eBFP2 (1.UASTAL14mKate 20 fmoles 61 5. (B + R)₂UASTAL14mKate 20 fmoles 61

7. (B + R + GAL)₁

UASTAL14mKate hEF1aGal4VP16 8. (B + R + GAL)₂UASTAL14mKate hEF1aGal4VP16

10. (B + R + GAL)₁

UASTAL14mKate hEF1aGal4VP16 (1.5 fmoles)

11. (B + R + GAL)₂

TAL14 UASTAL14mKate hEF1aGal4VP16 hEF1a-TAL14 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. Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

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)

//////

6. Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

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

14.

No DNA

No Lipofectamine

Tuning L7ae Repression

//////

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
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), GGTRE-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:
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:
7.
pEXPR: hEF1a-rtTA
MAV1212-hEF1a-eBFP2 (1.0 5 MAV1212-TRE-L7ae
10X DOX
8MAV1212-10 5 5 100X 9MAV1212-10 5 5 1000X 10MAV1212-10 5 5 2000X
11.
Dummy DNA (1 ug = ___ uL)
With Lipofectamine
12.
Dummy DNA (1 ug = ___ 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 (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
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
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.
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)
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
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, 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 incomplete 7/29/14: (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:

3. miR-30d30d4 miR146a miR-146a
5. miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b 22 06 let-7fHigh Sensor let-7f 22 075
13.
pEXPR: hEF1a-mKate
Dummy DNA (5.1uL = 1.75 fmoles)22.
No DNA
No 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)
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)
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
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
RESULT: Brief description of experimental outcome (mean transfection efficiency and implications). Link to results page with data analysis.
L7ae Sensors (with siRNA)
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
GOAL: Test the sensors in the presence of siRNA
-1 incomplete 33 complete 7/26/14: Seed cells 34 incomplete 7/27/14: 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 incomplete 7/29/14: (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)___ 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:
1. miR-144
pEXPR: hEF1a-mKate
Low Sensor miR-144
Mature-144
Dummy DNA (___ uL = ___ fmoles)
2. miR-181c
pEXPR: hEF1a-mKate
Low Sensor miR-181c
Mature-181c
Dummy DNA (___ uL = ___ fmoles)
3. miR-30d
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miRlet-30d7f
MaturesiRNA-30d7f
Dummy DNA (___ 2.2 uL = ___ 0.75 fmoles)
4. miR-146a
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-146a
Mature-146a
Dummy DNA (___ uL = ___ fmoles)
//////
//////
////// ////// ////// //////
13.
pEXPR: hEF1a-mKate
Dummy DNA (5.1uL = 1.75 fmoles)
13. 5. miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b
Mature-125b(1.5 fmoles)
Dummy DNA (___ 3.6 uL = ___ 1.25 fmoles)
619. let-7f
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor let-7f
Mature-7f
Dummy DNA (___ uL = ___ fmoles)
1. miR-144
pEXPR: hEF1a-mKate
Low Sensor miR-144
Mature-144
Dummy DNA (___ uL = ___ fmoles)
2. miR-181c
pEXPR: hEF1a-mKate
Low Sensor miR-181c
Mature-181c
Dummy DNA (___ uL = ___ fmoles)
3. miR-30d
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-30d
Mature-30d
Dummy DNA (___ uL = ___ fmoles)
4. miR-146a
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-146a
Mature 146a
Dummy DNA (___ uL = ___ fmoles)
5. miR-125b
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor miR-125b
Mature-125b
Dummy DNA (___ uL = ___ fmoles)
6. let-7f
pEXPR: hEF1a-mKate
Wild Type CAG-2xKturn-eGFP
High Sensor let-7f
Mature-7f
Dummy DNA (___ uL = ___ fmoles)
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:

mKateWild Type CAG-2xKturn-eGFP (1.5 fmoles)___ ___ 22.
No DNA
No Lipofectamine
1.
Untransfected

2.
pEXPR: hEF1a-mKate
Low Sensor miR-144c
Dummy DNA (2.9 uL = 1 fmoles)

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

//////

//////

//////
//////
3.
13.
pEXPR: hEF1a-mKate
Low Sensor miR-144
Dummy DNA (___ 2.9 uL = ___ 1 fmoles)13
4.
pEXPR: hEF1a-
mKate
High Sensor miR-30d
Dummy DNA (
2.9 uL =
1 fmoles)5
195.
Dummy DNA
With Lipofectamine
20.
Dummy DNA
With Lipofectamine
21.
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
32 complete Day 1: Seed cells 9 complete 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

Toggle Cloak

Treatment

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

A3

Hef1a: eBFP 250ng

TRE: miRNA1-mKATE250ng

Hef1a: rtTA 250ng

Dummy DNA 250ng
0 nM Dox

A4

Hef1a: eBFP 250ng

TRE: miRNA1

Cloak

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

A1 Hef1a: eBFP *250*ng 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 250*ng 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

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

- -mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

0 nM Dox

A5

A9

- Hef1a: eBFP 250ng
- TRE:

miRNA1

- miRNA3-mKATE

250ngDummy DNA

- 250ng
- Hef1a: rtTA

250ng

- 250ng
- Hef1a: BACE1

- 250ng

1000 nM Dox

A6

A10

- Hef1a: eBFP 250ng
- TRE:

miRNA1

- miRNA4-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

1000

0 nM Dox

A7

A11

- Hef1a: eBFP 250ng
- TRE: miRNA4-mKATE 250ng
- Hef1a:

BFP

- rtTA 250ng

Dummy DNA 500ng

Testing inducible expression of BACE2

- 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

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