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SAMPLE

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Sample Experiment #1 (Template)

4 incomplete Day 1: Seed cells 5 incomplete Day 2: Transfect Promoter:Gene1 (___ ng/uL - transfection marker), Promoter:Gene2 (___ ng/uL) and Promoter:Gene3 (___ ng/uL) into HEK cells 8 incomplete Day 3: Add DOX (if necessary) 6 incomplete 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)

36 complete 7/3: Seed cells 23 complete 7/4: Transfect hEF1a:eYFP (472 ng/uL) and hEF1a:tagBFP (1176 ng/uL) into HEK cells 24 complete 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.

...

Toggle Cloak
miRNA Detection

 

 

Neg Ctrl

    • Dummy DNA
    • With Lipofectamine

Neg Ctrl

    • Dummy DNA
    • With Lipofectamine

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

Cloak

Experiment #1L7ae Repression

Attempt #1

GOAL: Test the strength of L7ae

3 complete 7/14/14: Seed cells 22 complete 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 0 complete 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 Inconclusive. No L7ae was added.


Attempt #2

2 complete 7/16/14: Seed cells 1 complete 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 complete 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 Inconclusive: Too much DNA in each well.


Attempt #3

17 complete 7/24/14: Seed cells 18 complete 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 19 complete 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)21

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)12

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)12

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 Data is available

 

Experiment #2L7ae Tuning

GOAL: Test the strength sensitivity of TAL14L7ae repression

14 complete 7/25/1421 complete Day One: Seed cells 15 complete 7/26/14 28 complete Day Two: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, Wild Type hEF1a-2xKturn-eGFP, MAV1212-hEF1a-GAL4VP16L7ae, pEXPR: hEF1a-TAL14, and hEF1a-PirB rtTA (169.0 ng/uL), GG-Donor (dummy DNA) all at ~500 ng/uL into HEK cells 16 incomplete 7/28/14 (4 pm 29 complete Day Three: DOX Induction 30 complete Day Four (___ am): Flow Cytometry
     
    • DNA Dilution: MAV1212-hEF1a-eBFP2 (0.47 uL), TAL14-mKate Wild Type CAG-2xKturn-eGFP (0.62 96 uL), hEF1aMAV1212-TRE-GAL4VP16 L7ae (0.72 89 uL), pEXPR: hEF1a-TAL14 (___ rtTA (0.27 uL), Dummy DNA GG-Donor (to 1 ug)

    24-WELL PLATE TRANSFECTION MAP:

     (B)125 fmoles 076 (B)225 fmoles 076

    3. Neg Ctrl

    Dummy DNA (.2 fmoles = 205 uL)

    4. (B + R)1

    With Lipofectamine

    UASTAL14mKate 20 fmoles 61 5. (B + R)2UASTAL14mKate 20 fmoles 61 //////6

    7. (B + R + GAL)1

    Neg Ctrl

    Dummy DNA (3.2 fmoles = 2.05 uL)

    With Lipofectamine

    UASTAL14mKate hEF1aGal4VP16 8. (B + R + GAL)2UASTAL14mKate hEF1aGal4VP16

    10. (B + R + GAL)1

    UASTAL14mKate hEF1aGal4VP16 hEF1a-TAL14 11. (B + R + GAL)2UASTAL14mKate hEF1aGal4VP16 -TAL14 12. Neg/Neg Ctrl

    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)

    //////

    //////

     

    		//////

    9. Neg/Neg Ctrl

    No DNA

    No Lipofectamine

    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

    Experiment #3

    14.

    No DNA

    No Lipofectamine

     

    //////

     

    //////


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

     L7ae Quantification

    GOAL: Use TASBE tools to quantify the repression of L7ae

    32 complete 8/7/

    GOAL: Test the sensors in absence of siRNA

    10 incomplete 7/26/14: Seed cells 11 incomplete 7/279 complete 8/8/14: Transfect Low Sensor miRhEF1a-144 rtTA (708200.2 0 ng/uL), Low Sensor miRTRE-181c (628.1 L7ae (200 ng/uL), Wild Type 2x-Kturn eGFP (200 ng/uL), TRE-mKate (200.0 ng/ uL), High Sensor miRhEF1a-30d eBFP (1046200.2 0 ng/uL), High Sensor miRhEF1a-146a mKate (828200.8 0 ng/uL), High Sensor 125b (1159.5 hEF1a-eYFP (200.0 ng/uL) and High Sensor let-7f (889.6 , and GG Donor (200 ng/uL) into HEK cells 12 incomplete 7/2931 complete 8/10/14: (4 2 pm): Flow Cytometer

     

    • DNA Dilution: Low Sensor miRhEF1a-144 (___ rtTA (0.60 uL), TRE-L7ae (1.48 uL), Low Sensor miR-181c (___ Wild Type 2x-Kturn eGFP (1.19 uL), High Sensor miRTRE-30d (___ mKate (1.0 uL), High Sensor miRhEF1a-146a (___ eBFP (0.93 uL), High Sensor 125b (___ hEF1a-mKate (0.93 uL), High Sensor lethEF1a-7f (___ eYFP (0.93 uL)

    24-WELL PLATE TRANSFECTION MAP:

    miR-144

    Low Sensor miR-144 ()

    3. miR-30d

    High Sensor miR-30d

    4. miR-146a

    High Sensor miR-146a 5 High Sensor miR-125b

    5. miR-125b

    6 let-7fHigh Sensor let-7f 5 Low Sensor miR-144

    7. miR-144

    miR-181cLow Sensor miR-181c 5

    9. miR-30d

    High Sensor miR-30d 5

    12. let-7f

    High Sensor let-7f )

    Neg Ctrl

    Dummy DNA (3.2 fmoles = 2.05 uL)

    With Lipofectamine

    Neg Ctrl

    Dummy DNA (3.2 fmoles = 2.05 uL)

    With Lipofectamine

    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

    2. miR-181c

    Low Sensor miR-181c (1.5 fmoles)

    (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)

    10. miR-146a

    High Sensor miR-146a (1.5 fmoles)

    11. miR-125b

    High Sensor miR-125b (1.5 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

    //////

    //////

    		////////////////////////

    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

    13 complete Day 1: Seed cells 9 incomplete 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 20 incomplete 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/Neg Ctrl

    • No DNA
    • No Lipofectamine

Neg/Neg Ctrl

    • No DNA
    • No Lipofectamine

 

//////

 

//////

Toggle Cloak
Treatment

= 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

 L7ae Sensors (without siRNA)

GOAL: Test the sensors in absence of siRNA

10 complete 8.11.14: Seed cells 11 complete 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 12 complete 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 (5.1uL = 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 .

 L7ae Sensors (with siRNA)

GOAL: Test the sensors in the presence of siRNA

-1 incomplete 33 complete 8.12.14: Seed cells 34 complete 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. 35 complete 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 (5.1uL = 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 .

L7ae Sensors (with and without siRNA)

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

-1 incomplete 14 incomplete Day 1: Seed cells 15 incomplete 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. 16 incomplete 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 (5.1uL = 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 .


 L7ae Sensors (Multi Input)

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

-1 incomplete 20 complete 9.23.14: Seed cells 42 complete 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. 43 complete 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 .

TAL14 Repression

GOAL: Test the strength of TAL14

44 complete 7/25/14: Seed cells 45 complete 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 46 complete 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

 TAL14 Tuning

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

26 complete Day One: Seed cells 27 complete Day Two: Transfect MAV1212-hEF1a-eBFP2, TAL14-mKate, hEF1a-GAL4VP16, hEF1a-TAL14, and hEF1a-PirB (dummy DNA) at ~500 ng/uL into HEK cells 25 complete 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.

Toggle Cloak
Treatment

Cloak

Testing effectiveness of miRNA1–miRNA4 in down-regulating BACE1 expression v1

A1

    • Hef1a: eBFP 250ng
    • 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 250ng
    • 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

Cloak
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

A4

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

0 nM Dox

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

     

    A5

      • Hef1a:
      • rtTA 333ng

    1 nM Dox

      Hef1a:
      • eBFP 250ng
      • TRE: miRNA1-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng
    1

    1000 nM Dox

    		 A6
      • Hef1a: eBFP
      • 333ng
      • 250ng
      • TRE:
      • miRNA1 333ng
      • miRNA2-mKATE 250ng
      • Hef1a: rtTA
      • 333ng
    10
      • 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

    A7

      • Hef1a: eBFP 250ng
      • TRE:
      • miRNA1
      • miRNA2-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng
    10

    1000 nM Dox

    A8

      • Hef1a: eBFP
      • 333ng
      • 250ng
      • TRE:
      • miRNA1 333ng
      • miRNA3-mKATE 250ng
      • Hef1a: rtTA
      • 333ng
    100
      • 250ng
      • Hef1a: BACE1 250ng

    0 nM Dox

     A9

      • Hef1a: eBFP 250ng
      • TRE:
      • miRNA1
      • miRNA3-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng
    100

    1000 nM Dox

    A10

      • Hef1a: eBFP
      • 333ngTRE: miRNA1 333ng
      • 250ng
      • TRE: miRNA4-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a:
      • rtTA 333ng
      • BACE1 250ng
    1000

    0 nM Dox

    A11

      • Hef1a: eBFP 250ng
      • TRE:
      • miRNA1
      • miRNA4-mKATE 250ng
      • Hef1a: rtTA 250ng
      • Hef1a: BACE1 250ng

    1000 nM Dox

    B1

      • Hef1a:eBFP 333ng
      • Dummy DNA 666ng

     

    Negative Control

    B2

      TRE: miRNA1
      • Hef1a:eBFP
      • 333ng
      • Hef1a:rtTA 333ng
      • TRE:BACE2 333ng
    2000

     

    0 nM Dox

    B3

      • Hef1a:eBFP
      • 250ngTRE: miRNA1 250ng
      • 333ng
      • Hef1a:rtTA
      • 250ng
      • 333ng
    • Hef1a: BACE1 250ng
      • TRE:BACE2 333ng

     

    1000 nM Dox

    		 B4
      • No DNA (untransfected)

     

    Double negative control

    		 

     

    		 

     

     2000 nM Dox