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

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(Procedure)
(Procedure)
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#Starter Cultures with 25mL water had .8793g, .8800g, .8748g, and .8757g broth in them.
#Starter Cultures with 25mL water had .8793g, .8800g, .8748g, and .8757g broth in them.
#Expression cultures with 1L water had 25.0289g, 25.0069g, 25.002g, and 25.0036g broth in them.
#Expression cultures with 1L water had 25.0289g, 25.0069g, 25.002g, and 25.0036g broth in them.
 +
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:25, 7 October 2012 (EDT)''':inidiacte how you distigushedthe flasks..1, 2, 3, 4? or A, B, C, D? how do you know if htey act differently due to the amounts of chemicals added?
#Amount of Kanamycin for cultures was calculated. Kanamycin is an antibiotic and is being used to prevent microbial growth besides that of E. Coli.
#Amount of Kanamycin for cultures was calculated. Kanamycin is an antibiotic and is being used to prevent microbial growth besides that of E. Coli.
##The molarity was converted to µg/mL and the equation M<sub>1</sub>V<sub>1</sub> = M<sub>2</sub>V<sub>2</sub> was used to calculate the volume of stock that should go in broth.
##The molarity was converted to µg/mL and the equation M<sub>1</sub>V<sub>1</sub> = M<sub>2</sub>V<sub>2</sub> was used to calculate the volume of stock that should go in broth.
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#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.
#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.
#The plasmid that codes for Adenosine Deaminase (ADA)was inserted into the broth.
#The plasmid that codes for Adenosine Deaminase (ADA)was inserted into the broth.
 +
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:25, 7 October 2012 (EDT)''':plasmid is in the e. coli, therefore you add a colony of E. coli cells that contain the plasmid for ADA. split?
#The flasks were placed in an orbital incubator shaker at 37°C and 237 rpm
#The flasks were placed in an orbital incubator shaker at 37°C and 237 rpm
 +
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:25, 7 October 2012 (EDT)''':not that this was left to shake overnight
==Discussion==
==Discussion==

Revision as of 11:25, 7 October 2012

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Objectives

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

Procedure

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. Autoclave
    1. The flasks were put in at 121°C for 60 minutes. Due to limited space, the flasks had to be autoclaved in two batches.
    2. 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.
  4. Starter Cultures with 25mL water had .8793g, .8800g, .8748g, and .8757g broth in them.
  5. Expression cultures with 1L water had 25.0289g, 25.0069g, 25.002g, and 25.0036g broth in them.
  • Abigail E. Miller 11:25, 7 October 2012 (EDT):inidiacte how you distigushedthe flasks..1, 2, 3, 4? or A, B, C, D? how do you know if htey act differently due to the amounts of chemicals added?
  1. Amount of Kanamycin for cultures was calculated. Kanamycin is an antibiotic and is being used to prevent microbial growth besides that of E. Coli.
    1. 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.
    2. 50mg/mL Kanamycin * 1µg/.001mg = 50,000µg/mL Kanamycin
    3. 50µg/mL * 35mL = 50000µg/mL * volume needed
    4. .035mL Kanamycin solution needed
    5. The antibiotic was allowed to warm to room temperature and then inserted to the broth. The remaining Kanamycin went back in the freezer.
  2. 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.
  3. The plasmid that codes for Adenosine Deaminase (ADA)was inserted into the broth.
  • Abigail E. Miller 11:25, 7 October 2012 (EDT):plasmid is in the e. coli, therefore you add a colony of E. coli cells that contain the plasmid for ADA. split?
  1. The flasks were placed in an orbital incubator shaker at 37°C and 237 rpm
  • Abigail E. Miller 11:25, 7 October 2012 (EDT):not that this was left to shake overnight

Discussion

  • ADA is being synthesised for upcoming experiments on it's potential ability to make gold into nanoparticles.



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