3.4 GFP-tags

3.4 GFP-tags kdorfman Tue, 09/25/2012 - 17:47

Live 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 - 17:56

Lipofectamine2000 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 - 21:03

Addgene 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

DNA concentration

DNA concentration kdorfman Fri, 10/01/2021 - 19:22
If your plasmid DNA is ng/uL (=ug/mL)
and you need ug DNA
useuL DNA solution