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SAMPLE

Sample Experiment #1 (Template)

Day 1: Seed cells
Day 2: Transfect Promoter:Gene₁ (___ ng/uL - transfection marker), Promoter:Gene₂ (___ ng/uL) and Promoter:Gene₃ (___ ng/uL) into HEK cells
Day 3: Add DOX (if necessary)
Day 4 (time): Flow Cytometer

DNA Dilution: __uL Promoter:Gene₁; __uL Promoter:Gene₂; __uL Promoter:Gene₃
(Eg)_x: add _:_:_
1. __uL Promoter:Gene₁-lipid complex
2. __uL Promoter:Gene₂-lipid complex
3. __uL Promoter:Gene₃-lipid complex

24-WELL PLATE TRANSFECTION MAP:

//////	//////	//////	//////	//////	//////
+ve control Promoter:Gene₁	+ve control Promoter:Gene₁	-ve control dummy DNA with Lipofectamine	-ve control dummy DNA with Lipofectamine	-ve/-ve control no DNA no Lipofectamine	-ve/-ve control no DNA no Lipofectamine
///EMPTY WELL///	//////	//////	//////	//////	//////
(Eg)₁ __uM DOX Promoter:Gene₁ Promoter:Gene₂ Promoter:Gene₃	(Eg)₂ __uM DOX Promoter:Gene₁ Promoter:Gene₂ Promoter:Gene₃	(Eg)₃ __uM DOX Promoter:Gene₁ Promoter:Gene₂ Promoter:Gene₃	(Eg)₄ __uM DOX Promoter:Gene₁ Promoter:Gene₂ Promoter:Gene₃	(Eg)₅ __uM DOX Promoter:Gene₁ Promoter:Gene₂ Promoter:Gene₃	(Eg)₆ __uM DOX Promoter:Gene₁ Promoter:Gene₂ Promoter:Gene₃

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

Sample Experiment #2

(This was Practice Experiment #2)

7/3: Seed cells
7/4: Transfect hEF1a:eYFP (472 ng/uL) and hEF1a:tagBFP (1176 ng/uL) into HEK cells
7/6 (1pm): Flow Cytometer

DNA Dilution: 10uL hEF1a:eYFP; 5uL hEF1a:tagBFP
(Y+B)_x: add 1:1 (25uL hEF1a:eYFP/tagBFP-lipid complex)

Y₁ hEF1a:eYFP	(Y+B)₁ hEF1a:eYFP hEF1a:tagBFP	//////	//////	//////	-ve control dummy DNA with Lipofectamine
Y₂ hEF1a:eYFP	(Y+B)₂ hEF1a:eYFP hEF1a:tagBFP	//////	//////	//////	-ve control dummy DNA with Lipofectamine
B₁ hEF1a:tagBFP	(Y+B)₃ hEF1a:eYFP hEF1a:tagBFP	//////	//////	//////	-ve/-ve control no DNA no Lipofectamine
B₂ hEF1a:tagBFP	(Y+B)₄ hEF1a:eYFP hEF1a:tagBFP	//////	//////	//////	-ve/-ve control no DNA no Lipofectamine

RESULT: Extremely low transfection efficiency (<10%). Repeat to improve co-transfection results.

Antibody Detection

Transfection 1: Transfecting TRE:Syk-TEVp for Western Blot to compare the levels of endogenous vs exogenous Syk and to compare levels of expression under different amounts of dox induction.

Concentrations of DNA:

Dummy	1230 ng/ul
hEF1a:eYFP	1045 ng/ul
hEF1a:rtTa	169.4 ng/ul
TRE:Syk-TEVp	871 ng/ul

Volumes of DNA added:

Well 3

Well 4

Wells 7-12

Dummy 0.81 ul

Dummy 0.49 ul

hEF1a:eYFP 0.49

hEF1a:eYFP 0.38 ul

hEF1a:rtTA 1.18 ul

TRE:Syk-TEVp 0.46 ul

Well 1 HEK293	Well 2 EMPTY (Would have been B cells)	Well 3 HEK293 Dummy DNA 1000 ng	Well 4 HEK293 hEF1a:eYFP 400 ng Dummy DNA 600 ng	Well 5 EMPTY	Well 6 EMPTY
Well 7 HEK293 hEF1a:eYFP 400 ng hEF1a: rtTA 200 ng TRE: Syk-TEVp 400 ng 0 nM Dox	Well 8 HEK293 hEF1a:eYFP 400 ng hEF1a: rtTA 200 ng TRE: Syk-TEVp 400 ng 1 nM Dox	Well 9 HEK293 hEF1a:eYFP 400 ng hEF1a: rtTA 200 ng TRE: Syk-TEVp 400 ng 10 nM Dox	Well 10 HEK293 hEF1a:eYFP 400 ng hEF1a: rtTA 200 ng TRE: Syk-TEVp 400 ng 100 nM Dox	Well 11 HEK293 hEF1a:eYFP 400 ng hEF1a: rtTA 200 ng TRE: Syk-TEVp 400 ng 1000 nM Dox	Well 12 HEK293 hEF1a:eYFP 400 ng hEF1a: rtTA 200 ng TRE: Syk-TEVp 400 ng 2000 nM Dox

Receptor Detection

miRNA Detection

L7ae Repression

Attempt #1

GOAL: Test the strength of L7ae

7/14/14: Seed cells
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
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: Inconclusive. No L7ae was added.

Attempt #2

7/16/14: Seed cells
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
7/19/14 (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: Inconclusive: Too much DNA in each well.

Attempt #3

7/24/14: Seed cells
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
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 ___%. Data is available here

L7ae Tuning

GOAL: Test the sensitivity of L7ae repression

Day One: Seed cells
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
Day Three: DOX Induction
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: Transfection efficiency ___%. Data can be found here.

L7ae Quantification

GOAL: Use TASBE tools to quantify the repression of L7ae

8/7/14: Seed cells
8/8/14: Transfect hEF1a-rtTA (200.0 ng/uL), TRE-L7ae (200 ng/uL), Wild Type 2x-Kturn eGFP (200 ng/uL), TRE-mKate (200.0 ng/ uL), hEF1a-eBFP (200.0 ng/uL), hEF1a-mKate (200.0 ng/uL), hEF1a-eYFP (200.0 ng/uL), and GG Donor (200 ng/uL) into HEK cells
8/10/14: (2 pm): Flow Cytometer

DNA Dilution: hEF1a-rtTA (0.60 uL), TRE-L7ae (1.48 uL), Wild Type 2x-Kturn eGFP (1.19 uL), TRE-mKate (1.0 uL), hEF1a-eBFP (0.93 uL), hEF1a-mKate (0.93 uL), hEF1a-eYFP (0.93 uL)

24-WELL PLATE TRANSFECTION MAP:

1. Neg Ctrl

Dummy DNA

With Lipofectamine

2. Neg Ctrl

Dummy DNA

With Lipofectamine

3. Neg/Neg Ctrl

No DNA

No Lipofectamine

4. Neg/Neg Ctrl

No DNA

No Lipofectamine

5.

pEXPR: hEF1a-eYFP (1.0 fmoles)

Dummy DNA (1.5 fmoles = 4.4 uL)

6.

pEXPR: hEF1a-mKate (1.0 fmoles)

Dummy DNA (1.5 fmoles = 4.4 uL)

7.

pEXPR: hEF1a-eBFP (1.0 fmoles)

Dummy DNA (1.5 fmoles = 4.4 uL)

8.

pEXPR: hEF1a-eYFP (1.0 fmoles)

pEXPR: hEF1a-mKate (1.0 fmoles)

pEXPR: hEF1a-eBFP (1.0 fmoles)

Dummy DNA (0.75 fmoles = 2.2 uL)

9.

hEF1a-eBFP2 (0.5 fmole)

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

Dummy DNA (1.5 fmoles = 2.4 uL)

10.

pEXPR: hEF1a-rtTA (1.0 fmoles)

hEF1a-eBFP2 (0.5 fmole)

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

MAV1212-TRE-L7ae (1.0 fmoles)

pEXPR: TRE-mKate (1.0 fmoles)

0 uM DOX

11.

pEXPR: hEF1a-rtTA (1.0 fmoles)

hEF1a-eBFP2 (0.5 fmole)

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

MAV1212-TRE-L7ae (1.0 fmoles)

pEXPR: TRE-mKate (1.0 fmoles)

1X DOX

12.

pEXPR: hEF1a-rtTA 1.0 fmoles)

hEF1a-eBFP2 (0.5 fmole)

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

MAV1212-TRE-L7ae (1.0 fmoles)

pEXPR: TRE-mKate (1.0 fmoles)

10X DOX

13.

pEXPR: hEF1a-rtTA (1.0 fmoles)

hEF1a-eBFP2 (0.5 fmole)

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

MAV1212-TRE-L7ae (1.0 fmoles)

pEXPR: TRE-mKate (1.0 fmoles)

100X DOX

14.

pEXPR: hEF1a-rtTA (1.0 fmoles)

hEF1a-eBFP2 (0.5 fmole)

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

MAV1212-TRE-L7ae (1.0 fmoles)

pEXPR: TRE-mKate (1.0 fmoles)

1000X DOX

15.

pEXPR: hEF1a-rtTA (1.0 fmoles)

hEF1a-eBFP2 (0.5 fmole)

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

MAV1212-TRE-L7ae (1.0 fmoles)

pEXPR: TRE-mKate (1.0 fmoles)

2000X DOX

RESULT: Transfection efficiency ___%. Data can be found here

L7ae Sensors (without siRNA)

GOAL: Test the sensors in absence of siRNA

8.11.14: Seed cells
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
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 (5.1 uL = 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 here.

L7ae Sensors (with siRNA)

GOAL: Test the sensors in the presence of siRNA

- 8.12.14: Seed cells
- 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.
- 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 (5.1 uL = 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 here.

L7ae Sensors (with and without siRNA)

GOAL: Test the sensors in the presence and absence of siRNA

- Day 1: Seed cells
- 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.
- 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 (5.1 uL = 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 here.

L7ae Sensors (Multi Input)

GOAL: Test the sensors in the presence and absence of siRNA

- 9.23.14: Seed cells
- 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.
- 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 here.

TAL14 Repression

GOAL: Test the strength of TAL14

7/25/14: Seed cells
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
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 here

TAL14 Tuning

GOAL: Determine how much TAL14 is needed to repress TAL14-UAS-mKate

Day One: Seed cells
Day Two: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB (dummy DNA) at ~500 ng/uL into HEK cells
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.

Treatment

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

0 nM Dox

A9

- Hef1a: eBFP 250ng
- TRE: miRNA3-mKATE 250ng
- Hef1a: rtTA 250ng
- Hef1a: BACE1 250ng

1000 nM Dox

A10

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

0 nM Dox

A11

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

1000 nM Dox

B1

- Hef1a:eBFP 333ng
- Dummy DNA 666ng

Negative Control

B2

- Hef1a:eBFP 333ng
- Hef1a:rtTA 333ng
- TRE:BACE2 333ng

0 nM Dox

B3

- Hef1a:eBFP 333ng
- Hef1a:rtTA 333ng
- TRE:BACE2 333ng

1000 nM Dox

B4

- No DNA (untransfected)

Double negative control