User:Michael F. Nagle/Notebook/Chem 571/2012/09/18

Objectives

• Grow E. Coli in LB broth, for the purpose of expressing ADA.

Procedure

1. Starter Culture and Expression Culture
   1. Starter culture mediaand expression culture media were made following procedure. 25mL water was used instead of 35mL in making the starter culture.
   2. The broth flasks were capped with foil.
   3. The flasks were put in the autoclave at 121°C for 60 minutes. Due to limited space, the flasks had to be autoclaved in two batches.
   4. The second round only went in for 50 minutes because the autoclave was still warm. After the autoclaving was complete for each round, the flasks were left in for 30 minutes while the autoclave depressurized. The pressure meter was confirmed to be at 0 before the door was opened.
   5. Starter Cultures with 25mL water had .8793g, .8800g, .8748g, and .8757g broth in them.
   6. Expression cultures with 1L water had 25.0289g, 25.0069g, 25.002g, and 25.0036g broth in them.
   7. Amount of Kanamycin for cultures was calculated. The molarity was converted to µg/mL and the equation M₁V₁ = M₂V₂ was used to calculate the volume of stock that should go in broth.
   8. 50mg/mL Kanamycin * 1µg/.001mg = 50,000µg/mL Kanamycin
   9. 50µg/mL * 35mL = 50000µg/mL
   10. .035mL Kanamycin
   11. The antibiotic was allowed to warm to room temperature and then inserted to the broth. The remaining Kanamycin went back in the freezer.
   12. The foil caps on the broth flasks was sterilized with a bunsen burner after each time they were removed and placed back on the flasks. Metal forceps, which were also sterilized with flame between uses, were used to pick up a splint, which was used to pick up one colony of E. Coli and transfer it to the broth. A different split was used each time.
   13. The plasmid that codes for Adenosine Deaminase (ADA)was inserted into the broth.
   14. The flasks were placed in an orbital incubator shaker at 37°C and 237 rpm