Timp:M9Protocol

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M9 Minimal Media Formulation for E.coli

Media to keep E.Coli alive.

Materials

  • Sodium phosphate dibasic - Sigma S5136-500g
  • Potassium phosphate monobasic - Sigma P5655-500g
  • Ammonium Chloride - Sigma A0171-100g
  • Sodium Chloride - Sigma S5886-500g
  • Magnesium sulfate - Sigma M2643-500g
  • Glycerol - Sigma G2025-500mL
  • Thiamine Hydrochloride - T1270-100g
  • Casamino Acids(500g) - Difco - 223120
  • Calcium Chloride - Sigma C5670-100g
  • Isopropyl B-D-1-thiogalactopyranoside (IPTG) - Sigma I5502-1g
  • Ampicillin ready made- Sigma A5354-10ml
  • Kanamyacin solution from Kanamyacin Sulfate- Shelton Scientific IB02120
  • 50ml Self-Standing Centrifuge Tube (Corning 430897)


Preparation of stock solutions

  • Note: These have decent shelf life – store at RT
  1. 5X M9 Salts
    • 1 L 18Mohm water
    • 30 g Na2HPO4
    • 15 g KH2PO4
    • 5 g NH4Cl
    • 2.5 g NaCl
    • Autoclave – this has a long shelf life
  2. Sterile Water
    • Autoclave 1 L 18Mohm water
  3. 20% Glycerol
    • 10 mL Glycerol
    • 40 mL 18Mohm water
    • Filter sterilize – .22 um filter
  4. 20% Casamino Acids
    • 5 g Casamino
    • 25 mL 18M water
    • Filter Sterilze
  5. 1 M MgSO4
    • 12.324 g MgSO4
    • 50 mL 18Mohm water
    • Filter Sterilze
  6. 200 mM Thiamine Hydrochloride
    • 3.3727 g Thiamine Hydrochloride
    • 50 mL 18M water
    • Filter Sterilize
  7. 100 mM CaCl2
    • 735 mg CaCl2
    • 50 mL 18M water
    • Filter Sterilze
  8. Kanamycin stock
    • 50 mg Kanamycin
    • 1 mL sterile water

How to prepare 50ml of M9 Media

  1. 10 mL 5X M9 Salts
  2. 50 uL 1 M MgSO4
  3. 50 uL Thiamine
  4. 500 uL 20% Glycerol
  5. 500 uL 20% Casamino
  6. 50 uL CaCl2
  7. 100 uL Kanamycin (or Ampicllin)
  • Note: For SM9 media do not add Thiamine and Casamino. SM9 is used to achieve gene ex pression while slowing down the cell division. Another advantage of SM9 is that lack of large Kasamino and Thiamine molecules will result in better gel integrity
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