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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 _:_:_
- __uL Promoter:Gene1-lipid complex
- __uL Promoter:Gene2-lipid complex
- __uL Promoter:Gene3-lipid complex
24-WELL PLATE TRANSFECTION MAP:
+ve control
| +ve control
| -ve control
| -ve control
| -ve/-ve control
| -ve/-ve control
|
| ///EMPTY WELL/// | ////// | ////// | ////// | ////// | ////// |
| ////// | ////// | ////// | ////// | ////// | ////// |
|---|---|---|---|---|---|
(Eg)1
| (Eg)2
| (Eg)3
| (Eg)4
| (Eg)5
| (Eg)6
|
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
| (Y+B)1
| ////// | ////// | ////// | -ve control
|
Y2
| (Y+B)2
| ////// | ////// | ////// | -ve control
|
B1
| (Y+B)3
| ////// | ////// | ////// | -ve/-ve control
|
B2
| (Y+B)4
| ////// | ////// | ////// | -ve/-ve control
|
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
| (B)2
| ////// | ////// | ////// | ////// |
(GM)1
| (GM)2
| (GM + B)1
| (GM + B)2
| (GM + B + L)1
| (GM + B + L)2
|
(GWT)1
| (GWT)2
| (GWT + B)1
| (GWT + B)2
| (GWT + B + L)1
| (GWT + B + L)2
|
Neg Ctrl
| Neg Ctrl
| Neg/Neg Ctrl
| Neg/Neg Ctrl
| ////// | ////// |
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
| (B)2
| ////// | ////// | ////// | ////// |
(GM)1
| (GM)2
| (GM + B)1
| (GM + B)2
| (GM + B + L)1
| (GM + B + L)2
|
(GWT)1
| (GWT)2
| (GWT + B)1
| (GWT + B)2
| (GWT + B + L)1
| (GWT + B + L)2
|
Neg Ctrl
| Neg Ctrl
| Neg/Neg Ctrl
| Neg/Neg Ctrl
| ////// | ////// |
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. (GWT + B)1 MAV1212-hEF1a-eBFP2 (1.0 fmole) Wild Type CAG-2xKturn-eGFP (1.5 fmoles) hEF1a-PirB (1.0 fmole = 0.99 uL) | 4. (GWT + B)2 MAV1212-hEF1a-eBFP2 (1.0 fmole) Wild Type CAG-2xKturn-eGFP (1.5 fmoles) hEF1a-PirB (1.0 fmole = 0.99 uL) | 5. (GWT + B + L)1 MAV1212-hEF1a-eBFP2 (1.0 fmole) Wild Type CAG-2xKturn-eGFP (1.5 fmoles) MAV1212-hEF1a-L7ae (1.5 fmoles) | 6. (GWT + B + L)2 MAV1212-hEF1a-eBFP2 (1.0 fmole) Wild Type CAG-2xKturn-eGFP (1.5 fmoles) MAV1212-hEF1a-L7ae (1.5 fmoles) | ////// | ////// |
7. (GM + B)1 MAV1212-hEF1a-eBFP2 (1.0 fmole) Multant CAG-2xKturn-eGFP (1.5 fmoles) hEF1a-PirB (1.0 fmole = 0.99 uL) | 8. (GM + B)2 MAV1212-hEF1a-eBFP2 (1.0 fmole) Multant CAG-2xKturn-eGFP (1.5 fmoles) hEF1a-PirB (1.0 fmole = 0.99 uL) | 9. (GM + B + L)1 MAV1212-hEF1a-eBFP2 (1.0 fmole) Multant CAG-2xKturn-eGFP (1.5 fmoles) MAV1212-hEF1a-L7ae (1.5 fmoles) | 10. (GM + B + L)2 MAV1212-hEF1a-eBFP2 (1.0 fmole) Multant CAG-2xKturn-eGFP (1.5 fmoles) MAV1212-hEF1a-L7ae (1.5 fmoles) | ////// | ////// |
11. Neg Ctrl hEF1a-PirB (3.2 fmoles = 2.05 uL) With Lipofectamine | 12. Neg Ctrl hEF1a-PirB (3.2 fmoles = 2.05 uL) With Lipofectamine | 13. Neg/Neg Ctrl No DNA No Lipofectamine | 14. Neg/Neg Ctrl No DNA No Lipofectamine | ////// | ////// |
RESULT: Transfection efficiency ___%. 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 (from Jeremy Gam)
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 (1.19 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 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-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.
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
| A2
Negative Control | A3
| A4
0 nM Dox | A5
| A6
1000 nM Dox |
A7
| |||||
Testing inducible expression of BACE2
B1
Negative Control | B2
0 nM Dox | B3
1000 nM Dox | B4
Double negative control | ||
Transfection: Week of 8/11
A1
Negative BACE1 Control | A2
Positive BACE1 Control | A3
mKATE control | A4
miRNA1g control | A5
| A6
0 nM Dox |
A7
| A8
1000 nM Dox | A9
| A10
0 nM Dox | A11
| A12
1000 nM Dox |
A13
| A14
0 nM Dox | A15
| A16
1000 nM Dox | A17
| A18
0 nM Dox |
A19
| A20
1000 nM Dox | B1
Negative Control | B2
0 nM Dox | B3
1000 nM Dox | B4
Double negative control |
Transfection 9/17
A1
Negative BACE1 Control | A2
Positive BACE1 Control | A3
mKATE control | A4
0 nM Dox
| A8
1000 nM Dox | A10
0 nM Dox |
A12
1000 nM Dox |
|
|
|
| |
A13
| A14
0 nM Dox | A15
| A16
1000 nM Dox | A17
| A18
0 nM Dox |
A19
| A20
1000 nM Dox | B1
Negative Control | B2
0 nM Dox | B3
1000 nM Dox | B4
Double negative control |
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