User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/18

Day 5

• Protein Expressions and Starters

Goals

• Grow E.Coli cells and have them express ADA protein.
• Follow Protocols for making starter and expression cultures of the cells.

Procedure

• [Started Culture Protocol]
  • add 35 mL H₂Oto each to 4 250mL erlenmeyer flask
  • LB was added to each flask.
    • Flask 1=.8793g
    • Flask 2=.8800g
    • Flask 3=.8748g
    • Flask 4=.8757g

(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)

• [Expression Culture Media]
  • 1L of distilled water were added to 4.8L Fernbach flasks
  • LB was added to each flask
    • Flask 1= 25.0289g
    • Flask 2= 25.0069g
    • Flask 3= 25.0020g
    • Flask 4= 25.0036g

(note that while there were the amounts measured out- the amount of LB added to the flasks was actually less due to loss of sample in transferring it from the weigh dish to the flask)

• Next, two of the erlenmeyer flasks and two of the Fernbach flasks were placed in the Autoclave at a temperature of 121°C for 60minutes followed by a 30 minute cool down period. After which, the other four samples were placed in the autoclave for 50 minutes at the same temperature and were cooled down for 30 minutes as well. The autoclave is used in order to kill all other living organisms so that they do not react with the LB because we want to only grow/culture the E.Coli cells.

• The antibiotic Kanamycin was added into each of the flasks. The stock solution of the antibiotic was 50mg/mL. The desired concentration of kanamycin for the flasks was 50μg/mL.

Using the formula M₁V₁=M₂V₂ The volume or amount of kanamycin that would need to go into each of the flasks was 50 μg/mL x 35mL ÷50,000 μg = .35mL Kanamycin.