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l 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
Experiment #1
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: 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
| (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: 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. (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) | 4. (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) | 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)1 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 ___%. FACS data is available here
Experiment #2
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 (___ uL), hEF1a-GAL4VP16 (___ uL), hEF1a-TAL14 (___ uL), hEF1a-PirB (to 1 ug)
24-WELL PLATE TRANSFECTION MAP:
1. (B)1 MAV1212-hEF1a-eBFP2 (1.0 fmole) | 2. (B)2 MAV1212-hEF1a-eBFP2 (1.0 fmole) | ////// | ////// | //////
| 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) | 5. (B + R)2 MAV1212-hEF1a-eBFP2 (1.0 fmole) UAS-TAL14-mKate (1.5 fmoles) | ////// | ////// | ////// | 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 |
Experiment #3
GOAL: Test the sensors in absence of siRNA
- 7/26/14: Seed cells
- 7/27/14: Transfect Low Sensor miR-144 (___ ng/uL), Low Sensor miR-181c (___ ng/uL), High Sensor miR-30d (___ ng/uL), High Sensor miR-146a (___ ng/uL), High Sensor 125b (___ ng/uL) and High Sensor let-7f (___ 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 (1.5 fmoles) | 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.
Experiment #4
GOAL: Test the sensors in the presence of siRNA
- Day 1: Seed cells
- Day 2: Transfect Low Sensor miR-144 (___ ng/uL), Low Sensor miR-181c (___ ng/uL), High Sensor miR-30d (___ ng/uL), High Sensor miR-146a (___ ng/uL), High Sensor 125b (___ ng/uL), High Sensor let-7f (___ 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
|
|
|
|
|
|
|
|
|
|
|
|
////// | ////// | ////// | ////// | ////// | ////// |
Neg Ctrl
| Neg Ctrl
| Neg/Neg Ctrl
| Neg/Neg Ctrl
|
////// |
////// |
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)
|
Positive Control |
| |||
0 nM Dox |
0 nM Dox |
1 nM Dox |
1 nM Dox |
10 nM Dox |
10 nM Dox |
100 nM Dox |
100 nM Dox |
1000 nM Dox |
1000 nM Dox |
2000 nM Dox |
2000 nM Dox |
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