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TASK | WhoDunnit | Observed |
---|
Split cells | AG, CR, LA, SS | AW |
Seed plates | AG, CR, LA, SS | AW |
Transfection | AG, LA | AC, SS |
FACs Prep | LA | CR |
Imaging (FloCyto) | BT | AG, AW, CR, JX, LA |
Practice Transfections:
CONTROLS:
- -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)
PRACTICE #1: Fluorescent protein under constitutive promoter
- 6/23: Seed cells
- 6/24: Transfect hEF1a:eYFP into HEK cells
- 6/26 (4.30pm): Flow Cytometer
- Determine optimal Lipofectamine LTX volume (24 well)
- Determine transfection efficiency (quantitatively)
PRACTICE #2: Co-transfection (2 fluorescent proteins under constitutive promoter)
- 7/2: Seed cells
- 7/3: Transfect hEF1a:eYFP and hEF1a:mKate into HEK cells
- 7/5 (__pm): Flow Cytometer
Y1
- _uL complete media
- _uL hEF1a:eYFP
- _uL Lipofectamine LTX
| (Y+R)1
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve control |
Y2
- _uL complete media
- _uL hEF1a:eYFP
- _uL Lipofectamine LTX
| (Y+R)2
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve control |
R1
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| (Y+R)3
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve/-ve control |
R2
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| (Y+R)4
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve/-ve control |
PRACTICE #3: Fluorescent protein under inducible promoter
- 7/_: Seed cells
- 7/_: Transfect TRE:mKate, hEF1a:rtTA and hEF1a:eYFP (transfection marker) into HEK cells
- 7/_: Add DOX
- 7/_ (__pm): Flow Cytometer
Y1
- _uL complete media
- _uL hEF1a:eYFP
- _uL Lipofectamine LTX
| (Y+R)1
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve control |
Y2
- _uL complete media
- _uL hEF1a:eYFP
- _uL Lipofectamine LTX
| (Y+R)2
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve control |
R1
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| (Y+R)3
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve/-ve control |
R2
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| (Y+R)4
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve/-ve control |
PRACTICE #4**: Co-transfection (2 fluorescent proteins under constitutive & inducible promoters)
- 7/_: Seed cells
- 7/_: Transfect hEF1a:eYFP, TRE:mKate and hEF1a:rtTA** into HEK cells
- 7/_: Add DOX
- 7/_ (__pm): Flow Cytometer
Y1
- _uL complete media
- _uL hEF1a:eYFP
- _uL Lipofectamine LTX
| (Y+R)1
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve control |
Y2
- _uL complete media
- _uL hEF1a:eYFP
- _uL Lipofectamine LTX
| (Y+R)2
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve control |
R1
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| (Y+R)3
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve/-ve control |
R2
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| (Y+R)4
- _uL complete media
- _uL hEF1a:mKate
- _uL Lipofectamine LTX
| ////// | ////// | ////// | -ve/-ve control |