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| TASK | WhoDunnit | Observed |
|---|
| Split cells | AG, CR, GB, JA, LA, SS | AW |
| Seed plates | AG, CR, GB, LA, SS | AW |
| Transfection | AG, GB, LA | AC, SS |
| FACS Prep | AG, LA | CR |
| Imaging (FloCyto) | BT | AG, AW, CR, JX, LA |
Practice Transfections:
CONTROLS
- +ve: single color transfection (eYFP, eBFP or mKate, as appropriate)
- +ve/+ve: tri-color control (eYFP, eBFP and mKate co-transfected: allows for comparison of color/intensity when imaging)
- -ve: perform protocol, excluding DNA, or with dummy DNA
- -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 | (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
- 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 | (R+B)1 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
|
| R2 | (R+B)2 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
|
| B1 | (R+B)3 | ////// | ////// | ////// | -ve/-ve control |
| B2 | (R+B)4 | ////// | ////// | ////// | -ve/-ve control |
RESULT: Efficiency ~20%
PRACTICE #3: Fluorescent protein under inducible promoter
- 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
- 20uL hEF1a:eYFP-lipid complex
- 10uL hEF1a:rtTA-lipid complex
- 20uL TRE:mKate-lipid complex
| +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
- 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
