User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/23

Continuing Expressions

1. Centrifuge the cultures grown overnight at 3500rpm at 4°C for 15 minutes.
2. Resuspend each of the pellets in 1mL of LB broth.
3. Add each of the resuspended pellets with broth to one of the four 2800mL flasks made and autoclaved yesterday with 1L of sterile LB.
4. Add 1mL 100mg/mL ampicillin to each flask.
   • 100mg/mL ampicillin prepared with 5mL of distilled water and 0.5g of ampicillin, mixed and sterile filtered.
5. Incubate these flasks at 37°C at 165rpm until OD₆₀₀=1.2.
6. Add 1mL mixture of 0.1M IPTG, 1M FeCl₃, 0.21M alpha aminolevulinic acid to each flask. Continue to incubate at 37°C overnight.
   • Mixture prepared: 1.5g FeCl₃ in 9mL dH₂O, let cool. Add 0.22g IPTG and 0.25 alpha aminolevulinic acid to the solution.