Team:CU Boulder/Protocols/Media


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Making growth media


LB (1L) SOC (1L)
Yeast Extract 5g Yeast Extract 5g
Tryptone or Peptone10g Tryptone 20g
NaCl 10g NaCl 0.5g
Agar (plates only) 15g MgSO4 4.8g
Dextrose 3.6g
KCl 0.186g

Tricks on making plates

  • Do not autoclave antibiotics or other heat sensitive ingredients
    • Let autoclaved media cool first
  • For variety of selection plates, divide media into 50 mL conicals
    • Add antibiotics to conicals, mix, then pour
  • Amounts
    • 500 mL makes slightly less than 1 sleeve
    • 50 mL makes 2 thick plates
  • Media can be autoclaved then let sit to harden for latter use
    • Microwave (on low) until completely melted
    • Recommended to use conical method
    • Otherwise, pour ONLY into sterile containers

Adding antibiotics

  • Do not autoclave antibiotics
  • If making liquid media, antibiotics can be added at any point after the media cools.
  • If making plates, let media cool on a stir plate. Once cool enough add antibiotic, let mix for 30-60 seconds then pour.
    • Informal trick: It is about cool enough when you can hold your hand to the flask for 5 seconds (but obviously, don’t burn yourself)
  • Antibiotic usage:

    • AntibioticStock ConcentrationWorking ConcentrationRatio Antibiotic:media (for high concentrations)
    Ampicillin100 mg/mL50-100 ug/mL1ul:1mL
    Chloramphenicol34 mg/mL34-170 ug/mL5ul:1mL
    Kanamycin50 mg/mL50 ug/mL1ul:1mL
    Tetracycline5 mg/mL20 ug/mL4ul:1mL


Making antibiotic stock solutions

  • Ampicillin (100ng/mL)
    • Add 4g of Ampicillin powder to 40mL H20
  • Chlor (34 ng/mL)
    • Add 2.72g of Chloramphenicol sulfate to 80mL of 100% EtOH
  • Kanamycin (10mg/mL)
    • Add 0.8g of kanamycin powder to 80mL H2O
  • Tetracycline (5mg/mL)
    • Add 0.4g of tetracycline HCL to 80mL of 70% EtOH
Team:CU-Boulder - 2015.igem.org