Practice Experiments (for Tissue Culture)

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

• 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.

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)

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)

RESULT: Efficiency ~20%

• 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

RESULTS: Efficiency >50%