lSAMPLE

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:

Dummy1230 ng/ul
hEF1a:eYFP1045 ng/ul
hEF1a:rtTa169.4 ng/ul
TRE:Syk-TEVp871 ng/ul

Volumes of DNA added:

Well 3Well 4Wells 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

  • MAV1212-hEF1a-eBFP2

(B)2

  • MAV1212-hEF1a-eBFP2
////////////////////////

(GM)1

  • Mutant Hef1a-2xKturn-eGFP

(GM)2

  • Mutant Hef1a-2xKturn-eGFP

(GM + B)1

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B)2

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B + L)1

  • Mutant Hef1a-2xKturn-eGFP
  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B + L)2

  • Mutant Hef1a-2xKturn-eGFP
  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT)1

  • Wild Type Hef1a-2xKturn-eGFP

(GWT)2

  • Wild Type Hef1a-2xKturn-eGFP


(GWT + B)1

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B)2

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B + L)1

  • Wild Type Hef1a-2xKturn-eGFP
  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + 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
////////////////////////

(GM)1

  • Mutant Hef1a-2xKturn-eGFP

(GM)2

  • Mutant Hef1a-2xKturn-eGFP

(GM + B)1

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B)2

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GM + B + L)1

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2
  • MAV1212-hEF1a-L7ae

(GM + B + L)2

  • Mutant Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2
  • MAV1212-hEFa-L7ae

(GWT)1

  • Wild Type Hef1a-2xKturn-eGFP

(GWT)2

  • Wild Type Hef1a-2xKturn-eGFP


(GWT + B)1

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B)2

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2

(GWT + B + L)1

  • Wild Type Hef1a-2xKturn-eGFP
  • MAV1212-hEF1a-eBFP2
  • MAV1212-hEFa1-L7ae

(GWT + 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. (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 (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

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

    • 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 1000ng


    Negative Control
  • Hef1a: eBFP 500ng
  • Hef1a: BACE1 500ng

Positive Control

 

   
  • Hef1a: eBFP 333ng
  • TRE: miRNA1 333ng
  • Hef1a: rtTA 333ng

0 nM Dox

  • Hef1a: eBFP 250ng
  • TRE: miRNA1 250ng
  • Hef1a: rtTA 250ng
  • Hef1a: BACE1 250ng

0 nM Dox

  • Hef1a: eBFP 333ng
  • TRE: miRNA1 333ng
  • Hef1a: rtTA 333ng

1 nM Dox

  • Hef1a: eBFP 250ng
  • TRE: miRNA1 250ng
  • Hef1a: rtTA 250ng
  • Hef1a: BACE1 250ng

1 nM Dox

  • Hef1a: eBFP 333ng
  • TRE: miRNA1 333ng
  • Hef1a: rtTA 333ng

10 nM Dox

  • Hef1a: eBFP 250ng
  • TRE: miRNA1 250ng
  • Hef1a: rtTA 250ng
  • Hef1a: BACE1 250ng

10 nM Dox

  • Hef1a: eBFP 333ng
  • TRE: miRNA1 333ng
  • Hef1a: rtTA 333ng

100 nM Dox

  • Hef1a: eBFP 250ng
  • TRE: miRNA1 250ng
  • Hef1a: rtTA 250ng
  • Hef1a: BACE1 250ng

100 nM Dox

  • Hef1a: eBFP 333ng
  • TRE: miRNA1 333ng
  • Hef1a: rtTA 333ng

1000 nM Dox

  • Hef1a: eBFP 250ng
  • TRE: miRNA1 250ng
  • Hef1a: rtTA 250ng
  • Hef1a: BACE1 250ng

1000 nM Dox

  • Hef1a: eBFP 333ng
  • TRE: miRNA1 333ng
  • Hef1a: rtTA 333ng

2000 nM Dox

  • Hef1a: eBFP 250ng
  • TRE: miRNA1 250ng
  • Hef1a: rtTA 250ng
  • Hef1a: BACE1 250ng

2000 nM Dox