Plasmids

Submitted by kdorfman on 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
Promega midiprep manual
Addgene plasmid concs