TASKWhoDunnitObserved
Split cellsAG, CR, GB, JA, LA, SSAW
Seed platesAG, CR, GB, LA, SSAW
TransfectionAG, GB, LAAC, SS
FACS PrepAG, LACR
Imaging (FloCyto)BTAG, AW, CR, JX, LA

 

Practice Transfections:

CONTROLS

  1. +ve: single color transfection (eYFP, eBFP or mKate, as appropriate)
  2. +ve/+ve: tri-color control (eYFP, eBFP and mKate co-transfected: allows for comparison of color/intensity when imaging)
  3. -ve: perform protocol, excluding DNA, or with dummy DNA
  4. -ve/-ve: no actions performed on cells

PRACTICE #1: Fluorescent protein under constitutive promoter

  • 6/23: Seed cells
  • 6/24: Transfect hEF1a:eYFP (472 ng/uL) into HEK cells
  • 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.

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

ATTEMPT #1

  • 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.

 

ATTEMPT #2

  • 7/9: Seed cells
  • 7/10: Transfect hEF1a:mKate (621 ng/uL) and hEF1a:eBFP (405 ng/uL) into HEK cells
  • 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
//////////////////-ve control
  • dummy DNA
  • with Lipofectamine
R2
  • 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
//////////////////-ve/-ve control
  • no DNA
  • no Lipofectamine
B2
  • hEF1a:eBFP
(R+B)4
  • hEF1a:mKate
  • hEF1a:eBFP
//////////////////-ve/-ve control
  • no DNA
  • no Lipofectamine

RESULT: Efficiency ~20%

 

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

  • 7/10: Seed cells
  • 7/11: Transfect TRE:mKate (431 ng/uL), hEF1a:rtTA (169 ng/uL) and hEF1a:eYFP (1045 ng/uL - transfection marker) into HEK cells
  • 7/12: Add DOX
  • 7/13 (1:30pm): Flow Cytometer

 

  • 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