User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/03

Purpose

• Amplify ADA transfected BL21(DE3) supercompetitent E.coli cells using LB media

Procedure

• 4 sets of 35mL LB media from Teknova was made using the recipe below and placed in four separate 250mL Erlenmeyer flasks:

  0.88mg of LB broth media powder
  35mL of sterile water

• 4 sets of 1L LB media from Teknova was made using the recipe below and placed in four separate 2800mL Pyrex flasks:

  25g of LB broth media powder
  1L of sterile water

• 2 of 1L LB media and 2 of 35mL LB media were placed in autoclave. The autoclave goes through the following procedure for liquid cycles:
  • Water was filled to bottom of autoclave
  • Knob of autoclave was turned to STE
  • 2 of 1L LB media and 2 of 35mL of LB media were loaded into autoclave
  • Door was closed tightly
  • Timer was set to 60 minutes
  • After 60 minutes, the knob was left on STE for 30 minutes and until the pressure needle turned to 0
  • Door was opened and autoclaved LB media was unloaded
• The above procedure was repeated twice
• Oribital shaker was prewarmed to 37°C
• After cooling, 35uL of 0.4M Kanamycin was defrosted and pipetted into the four 250mL flasks including 35mL LB broth
• Plate labeled "ADA BL21(DE3)" was taken from fridge. Four cell colonies from the plate was picked out using sterile wooden sticks, and one cell colony from plate was dipped into each of 35mL LB broth
• Four 35mL LB medium with kanamycin and ADA transfected BL21(DE3) cell, making up the starter culture, was placed in orbital shaker for overnight (16 hours) for 37°C under 200rpm.

Notes

• When cell colonies were picked out from the plate, the process were performed under sterile environment to prevent contamination into LB media.
• Kanamycin was added into LB media as antibiotics to prevent the growth of non-ADA transfected cells inside the LB media
• The starter culture was left in shaker overnight for best cell population for cell amplification.