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

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

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 ___%. FACS data is available 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

Tuning L7ae Repression

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-hEF1a-L7ae (0.71 uL), pEXPR: hEF1a-rtTA (___ uL), GG-Donor (to 1 ug)

24-WELL PLATE TRANSFECTION MAP:

1. (B)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Dummy DNA (1.5 fmole = 1.60 uL)

2. (B)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

Dummy DNA (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)

Dummy DNA (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)

Dummy DNA (1.0 fmole = 0.99 uL)

5. (G_WT+ B + L)₁

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. (G_WT+ B + L)₂

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. (G_WT+ B + L)₃

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. (G_WT+ B + L)₄

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. (G_WT+ B + L)₅

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. (G_WT+ B + L)₆

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. Neg Ctrl

Dummy DNA (1 ug = ___ uL)

With Lipofectamine

12. Neg Ctrl

Dummy DNA (1 ug = ___ uL)

With Lipofectamine

13. Neg/Neg Ctrl

No DNA

No Lipofectamine

14. 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.

Tuning TAL14 Repression

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 (___ 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. (B + R)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

Dummy DNA (2.0 fmoles = 0.61 uL)

4. (B + R)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

Dummy DNA (2.0 fmoles = 0.61 uL)

5. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.5 fmoles)

6. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.5 fmoles)

10. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (1.0 fmoles)

11. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (1.5 fmoles)

10. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (2.0 fmoles)

11. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (2.5 fmoles)

10. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (3.0 fmoles)

11. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (3.5 fmoles)

10. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (4.0 fmoles)

11. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (4.5 fmoles)

10. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (5.0 fmoles)

11. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (10.0 fmoles)

10. (B + R + GAL)₁

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (15.0 fmoles)

11. (B + R + GAL)₂

MAV1212-hEF1a-eBFP2 (1.0 fmole)

UAS-TAL14-mKate (1.5 fmoles)

hEF1a-Gal4VP16 (1.0 fmoles)

hEF1a-TAL14 (20.0 fmoles)

12. Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

13. Neg Ctrl

Dummy DNA (3.2 fmoles = 2.05 uL)

With Lipofectamine

14. Neg/Neg Ctrl

No DNA

No Lipofectamine

15. 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 (without siRNA)

GOAL: Test the sensors in absence of siRNA

7/26/14: Seed cells
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
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

Day 1: Seed cells
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
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.

Treatment

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

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

Hef1a: eBFP *250*ng Dummy DNA 750ng Positive Control	Hef1a: eBFP *250ng Hef1a: BACE1 250ng Dummy DNA 500ng* Negative Control	Hef1a: eBFP *250ng TRE: miRNA1 250ng Hef1a: rtTA 250*ng Dummy DNA 250ng 0 nM Dox	Hef1a: eBFP 250ng TRE: miRNA1 250ng Hef1a: rtTA 250ng Hef1a: BACE1 250ng 0 nM Dox	Hef1a: eBFP *250ng* TRE: miRNA1 *250ng Hef1a: rtTA 250ng Dummy DNA 250ng* 1000 nM Dox	Hef1a: eBFP 250ng TRE: miRNA1 250ng Hef1a: rtTA 250ng Hef1a: BACE1 250ng 1000 nM Dox

Testing inducible expression of BACE2

Hef1a:eBFP 333ng Dummy DNA 666ng Negative Control	Hef1a:eBFP 333ng Hef1a:rtTA 333ng TRE:BACE2 333ng 0 nM Dox	Hef1a:eBFP 333ng Hef1a:rtTA 333ng TRE:BACE2 333ng 1000 nM Dox