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Common Media

Bacterial Media and Additives

LB Broth (1 L)

  • 1 L dH2O
  • 10 g Bacto  tryptone
  • 5 g Bacto  yeast extract
  • 5 g NaCl
  • 1 mL 2N NaOH
  • 12 g  agar  (optional)
    • 3 g agar + 250 mL broth per bottle

Autoclave
Note: Add 3 mL 1 M CaCl 2 before phage transductions

LB-Top agar (500 mL)

Note: Add 1.5 mL 1 M CaCl 2 before phage transductions

Swarm agar for

E. coli

(400 mL)

  • 400 mL dH2O
  • 4 g Tryptone
  • 2 g NaCl
  • 0.88 g Agar (for 0.22%)

Autoclave

TY broth (1 L)

  • 1 L dH2O
  • 6 g Bacto  tryptone
  • 3 g Bacto  yeast extract
  • 0.5 g CaCl2·2H2O (this is equal to 3.4 mM)
  • 12 g Bacto  agar  (optional)
    • 3 g agar + 250 mL broth per bottle

Autoclave
Note: for motility assays, make agar 0.22%

TY top agar (500 mL)

  • 500 mL dH2O
  • 3 g tryptone
  • 1.5 g yeast extract
  • 0.25 g CaCl2·2H2O (=3.4 mM)
  • 2 g agar

Autoclave
Note: You do not need to add any additional calcium when performing phage transductions

PH medium (1 L)

  • 1 L dH2O
  • 4 g peptone
  • 0.5 g yeast extract
  • 0.5 g tryptone
  • 0.5 g CaCl2·2H2O
  • 0.2 g MgSO4·7H2O
  • 12 g agar (optional)
    • 3 g agar + 250 mL broth per bottle

Autoclave

PSY medium (1 L)

  • 1 L dH2O
  • 20 g peptone
  • 20 g sucrose
  • 5 g yeast extract
  • 12 g agar (optional)
    • 3 g agar + 250 mL broth per bottle

Autoclave
NOTE: For culturing Bradyrhizobium japonicum, supplement with 0.1% arabinose

YEM Broth (1 L)

  • 1 L dH2O
  • 10 g mannitol
  • 1 g Bacto  yeast extract
  • 500 mg K2HPO4
  • 200 mg MgSO4·7H2O
  • 100 mg NaCl
  • 12 g  agar  (optional)
    • 3 g agar + 250 mL broth per bottle

Autoclave

Water

agar

(250 mL)

  • 250 mL dH2O
  • * 3 g  agar

Autoclave

Standard Minimal Medium (SMM) (250 mL)

To 250 mL sterile dH

2

O or

water agar

add:

Autoclave
Note: Very mucoid growth results from SMAJ at 1/2X—especially in sucrose

100X SMAJ (50 mL)

  • ~30 mL ddH2O
  • 8 g KH2PO4
  • 12 g K2HPO4
  • 2 g NaCl
  • 4 g NH4Cl

(takes a while to dissolve)
QS to volume with ddH2O
Filter sterilize
Store at room temperature

100X MCBT (50 mL)

  • 50 mL ddH2O
  • 2.46 g MgSO4·7H2O
  • 1 mg CoCl2
  • 1 mg  biotin
  • 3 mg  thiamine

Filter sterilize
Note: Biotin takes a while to dissolve
Store at room temperature

100X (30%) carbon source (50 mL)

Filter sterilize
Store at room temperature
Note: mannitol and lactose are soluble at 30% only upon heating

100X (2%) calcofluor (40 mL)

  • 0.8 g calcofluor
  • QS to volume with ddH2O

Filter sterilize
Store at room temperature

Plant Media and Additives


Standard Nodulation Medium (SNM) (1 L)

Agar slants

Microwave to dissolve agar
Distribute 8.2 mL per 18×150 disposable tube
Cap tubes
Autoclave for 10 minutes
Wet the tubes (this way they'll stick to the rack instead of sliding out) Slant the racks carefully, making sure the agar reaches the bottom of the cap Allow to cool for at least one hour and preferably overnight

Broth

  • 1 L dH2O
  • 0.35 g KH2PO4
  • pH to 7.0 (~850 μL 2 N  KOH)

Autoclave for 15 minutes
Add:

500X magnesium sulfate (50 mL)

  • 50 mL ddH2O
  • 6.25 g MgSO4·7H2O

Filter sterilize

500X calcium chloride (50 mL)

  • 50 mL ddH2O
  • 3.75 g CaCl2·2H2O

Filter sterilize

500X minor salts (200 mL)

  • 1.9 g Na2 EDTA ·2H2O
  • 1.4 g FeSO4·7H2O
  • 300 mg ZnSO4·7H2O
  • 300 mg H3BO3
  • 300 mg MnSO4·H2O
  • 30 mg Na2MoO4·2H2O
  • 3 mg CuSO4
  • 3 mg CoCl2

QS to volume with ddH2O
Some heating may be required to get it all into solution
Filter sterilize

500X Potassium nitrate (40 mL)

  • 40 mL ddH2O
  • 4.04 g potassium nitrate (KNO3)

Filter sterilize