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- -ve/-ve: no actions performed on cells
- -ve: perform protocol, excluding DNA, or with dummy DNA
- +ve: tri-color control (perform experiments with YFP, BFP, mKATE separately, and one with all 3: allows for comparison of color/intensity when imaging)
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PRACTICE #1: Fluorescent protein under constitutive promoter
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ATTEMPT #1 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 | (Y+B)1 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
| | Y2 | (Y+B)2 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
| | B1 | (Y+B)3 | ////// | ////// | ////// | -ve/-ve control | | B2 | (Y+B)4 | ////// | ////// | ////// | -ve/-ve control |
RESULT: Extremely low transfection efficiency (<10%). Repeat to improve co-transfection results. ATTEMPT #2 22 incomplete 7/_: Seed cells 37 incomplete 7/_: Transfect hEF1a:eYFP (___ ng/uL) and hEF1a:tagBFP (___ ng/uL) into HEK cells 38 incomplete 7/_ (_pm): Flow Cytometer - DNA Dilution: 10uL hEF1a:eYFP; 5uL hEF1a:tagBFP
- (Y+B)x: add 1:1 (25uL hEF1a:eYFP/tagBFP-lipid complex)
| Y1 | (Y+B)1 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
| | Y2 | (Y+B)2 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
| | B1 | (Y+B)3 | ////// | ////// | ////// | -ve/-ve control | | B2 | (Y+B)4 | ////// | ////// | ////// | -ve/-ve control |
RESULT: |
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