3.4 GFP-tags
3.4 GFP-tags kdorfman Tue, 09/25/2012 - 17:47Live cells on MatTek dishes:
Task 2. GFP-alpha-tubulin cells (everybody) 8 dishes
Task 3. Nucleofected with GFP version of (2 different ones per group) 4 - 8 dishes each:
- Rab11
- E cadherin
- H2B (GFP)
- alpha actinin
Lipofectamine
Lipofectamine kdorfman Tue, 09/25/2012 - 17:56Lipofectamine2000 Plasmid Transfection
24-48 hrs before class,
- transfect cells (at 40-60% confluency)
- Medium without
- antibiotic
- serum
- L-glutamine
Protocol
- Dilute DNA with DMEM
- Dilute lipofectamine2000 with DMEM, incubate <5 min at RT
- Mix DNA with Lipofectamine2000, incubate >15 min at RT
- Rinse 2x with DMEM
- Add complexes to cells, incubate 4 hr 37C
- Rinse 2x with warm PBS
Add normal medium back to cells
Per 35 mm dish (1 coverslip):
- 5 µg DNA in 500 µL
- 10 µL lipofectamine in 500 µL
Plasmids
Plasmids kdorfman Tue, 09/25/2012 - 21:03Addgene plasmids have G418 (neomycin) resistance except 26737
(E. coli for plasmids are in -80)
| gene | fluorophore | resistance | mg/mL (Abs 280) | mg/mL (260/280) | µL to get 2 µg | source |
|---|---|---|---|---|---|---|
| rab11 WT | GFP | kan | 6.3 | 7.8 | 0.6 | Addgene 12674 |
| E cadherin | EGFP | amp | 2.5 | 2.5 | 1 | PW |
| H2B | m cherry | kan | 1.8 | 1.3 | PW | |
| H2B | EGFP | kan | 5 | 7.3 | 0.33 | PW |
| EB1 | EGFP | kan | 0.662 | 0.86 | too low | PW |
| Actin | EGFP | kan | 5.2 | 4.97 | 0.4 | PW |
| a-actinin | EGFP | kan | 1.29 | 2.73 | 1 | PW |
| life act | GFP | kan (G418 for transfected selection - 1mg/mL for 3t3) | 99.1 ng/uL | 280: 1.054 | 260: 1.983 | Addgene 58470 |
| H2B | mCherry | Amp | Addgene 20972 | |||
| UtrCH | GFP | Amp | Addgene 26737 | |||
| E-cadherin | GFP | Amp | Addgene 28009 | |||
| Sec61 beta | mcherry | Kan | Addgene 49155 | |||
| Lifeact 7 | mcherry | Kan | Addgene 54491 | |||
| Golgi 7 | mcherry | Kan | Addgene 55052 | |||
| LaminA-C-18 | mcherry | Kan | Addgene 55068 | |||
| Lysosomes-20 | mcherry | Kan | Addgene 55073 | |||
| Mito-7 | DsRed2 | Kan | Addgene 55838 | |||
| Vimentin-7 | EGFP | Kan | Addgene 56439 | |||
| Sec61b-C1 | mEmerald | Kan | Addgene 90992 |
Making plasmids
PrepEase Endotoxin-Free Maxi Plasmid Kit, 78730 from USB
Streak cells on LB-30 µg/mL Kan or LB-amp 50 µg/mL (?)
Pick a single colony.
Inoculate 100 mL LB (+ 50 – 100 µg/mL ampicilin or 10 – 50 µg/mL kanamycin)
Shake overnight, 37C
Divide entire culture into 3 or 4 50-mL conicals
Use the appropriate rotor in the biochemistry floor model centrifuge.
Follow PrepEase directions, except: let filtrate drip directly into column (Steps 5-6)
Test in spectrophotometer at 1:500. Adjust dilution as needed
- Abs 260
- 260/280 ratio