Page History

TASK	WhoDunnit	Observed
Split cells	AG, CR, GB, JA, LA, SS	AW
Seed plates	AG, CR, GB, LA, SS	AW
Transfection	AG, GB, LA	AC, SS

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FACS Prep	AG, LA	CR
Imaging (FloCyto)	BT	AG, AW, CR, JX, LA

Practice Transfections:

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CONTROLS

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+ve

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: single color transfection (eYFP, eBFP or mKate, as appropriate)
+ve/+ve

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: tri-color control (

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eYFP, eBFP and mKate co-transfected: allows for comparison of color/intensity when imaging)
-ve: perform protocol, excluding DNA, or with dummy DNA
-ve/-ve: no actions performed on cells

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PRACTICE #1: Fluorescent protein under constitutive promoter

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11 complete 6/23: Seed cells 12 complete 6/24: Transfect hEF1a:eYFP (472 ng/uL) into HEK cells 13 complete 6/26 (4.30pm): Flow Cytometer Determine optimal Lipofectamine LTX volume (24 well) Determine transfection efficiency (quantitatively) *RESULT:* Decent transfection efficiency (~60%). Move on to Practice #2.

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PRACTICE #2: Co-transfection (2 fluorescent proteins under constitutive promoter)

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ATTEMPT #1

36

complete

7/

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3: Seed cells

23

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complete

7/

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4: Transfect hEF1a:eYFP (472 ng/uL) and hEF1a:

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tagBFP (1176 ng/uL) into HEK cells

24

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complete

7/

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6 (

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1pm): Flow Cytometer

PLATE:

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

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_uL complete media
_uL hEF1a:eYFP
_uL Lipofectamine LTX

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///	-ve/-ve control

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no DNA

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_uL complete media
_uL hEF1a:eYFP
_uL Lipofectamine LTX

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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.

ATTEMPT #2

22

complete

7/9: Seed cells

37

complete

7/10: Transfect hEF1a:mKate (621 ng/uL) and hEF1a:eBFP (405 ng/uL) into HEK cells

38

complete

7/12 (1:30pm): Flow Cytometer

DNA Dilution: 8.1uL (10 uL) hEF1a:mKate; 12.3uL (15 uL) hEF1a:eBFP
(R+B)_x: add 1:1 (25uL hEF1a:mKate/eBFP-lipid complex)

R₁

hEF1a:mKate

(R+B)₁

hEF1a:mKate
hEF1a:eBFP

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-ve control

dummy DNA

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with Lipofectamine

R₂

_uL complete media
_uL hEF1a:mKate
_uL Lipofectamine LTX

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hEF1a:mKate	(R+B)₂ hEF1a:mKate hEF1a:eBFP	//////	//////	//////	-ve control dummy DNA with Lipofectamine
B₁ hEF1a:eBFP	(R+B)₃ hEF1a:mKate hEF1a:eBFP	//////	//////	//////

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-ve/-ve control

cells (no action)

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no DNA no Lipofectamine
B₂ hEF1a:eBFP	(R+B)₄

hEF1a:mKate

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hEF1a:eBFP

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///	-ve/-ve control

cells (no action)

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no DNA
no Lipofectamine

RESULT: Efficiency ~20%

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PRACTICE #3: Fluorescent protein under inducible promoter (DOX ladder)

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14

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complete

7/

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10: Seed cells

15

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complete

7/

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11: Transfect TRE:mKate (431 ng/uL), hEF1a:rtTA (169 ng/uL) and hEF1a:eYFP (1045 ng/uL - transfection marker) into HEK cells

16

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complete

7/

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12: Add DOX

18

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complete

7/

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13 (1:30pm): Flow Cytometer

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DNA Dilution: 4.8uL hEF1a:eYFP; 29.6uL hEF1a:rtTA; 11.6uL TRE:mKate
Conc. of DOX: 1mg/uL
(D=x): add 2:1:2
1. 20uL hEF1a:eYFP-lipid complex
2. 10uL hEF1a:rtTA-lipid complex
3. 20uL TRE:mKate-lipid complex

+ve control hEF1a:eYFP	+ve control hEF1a:eYFP	-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
//////	//////	//////	//////	//////	//////
(D=0)₁ no DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	(D=0)₂ no DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	D=1 1mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	*D=2(3)** 2mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	*D=3(5)** 3mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	*D=4(10)** 4mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate
*D=5(2)** 5mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	*D=6(4)** 6mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	*D=7(6)** 7mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	*D=8(7)** 8mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	*D=9(8)** 9mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate	*D=10(9)** 10mg DOX hEF1a:eYFP hEF1a:rtTA TRE:mKate

RESULTS: Efficiency >50%

SUBGROUP-SPECIFIC PRACTICE EXPERIMENTS

Inducible and Constitutive Cascades

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Key

Practice Transfections: