You are viewing an old version of this page. View the current version.
Compare with Current
View Page History
« Previous
Version 41
Next »
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)
- 7/3: Seed cells
- 7/4: Transfect hEF1a:eYFP and hEF1a:mKate into HEK cells
- 7/6 (1pm): Flow Cytometer
Y1 | (Y+R)1 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
|
Y2 | (Y+R)2 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
|
R1 | (Y+R)3 | ////// | ////// | ////// | -ve/-ve control |
R2 | (Y+R)4 | ////// | ////// | ////// | -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
+ve control | +ve control | -ve control- dummy DNA
- with Lipofectamine
| -ve control- dummy DNA
- with Lipofectamine
| -ve/-ve control | -ve/-ve control |
////// | ////// | ////// | ////// | ////// | ////// |
(D=0)1- no DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=0.2
- 0.2uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=1- 1uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=1.5- 1.5uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=3- 3uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=4- 4uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
|
(D=0)2- no DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=6
- 6uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=7.5- 7.5uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=10- 10uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=17- 17uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=30- 30uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
|
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