Griffitts:Common media

LB Broth (1 L)
Autoclave Note: Add 3 mL 1 M CaCl2 before phage transductions
 * 1 L dH20
 * 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

LB-Top agar (500 mL)
Note: Add 1.5 mL 1 M CaCl2 before phage transductions
 * 500 mL dH20
 * 5 g Bacto tryptone
 * 2.5 g Bacto yeast extract
 * 2.5 g NaCl
 * 0.5 mL 2N NaOH
 * 2 g agar
 * Microwave until agar is dissolved
 * Distribute 75 mL per bottle
 * Autoclave

Swarm agar for E. coli (400 mL)
Autoclave
 * 400 mL dH20
 * 4 g Tryptone
 * 2 g NaCl
 * 0.88 g Agar (for 0.22%)

TY broth (1 L)
Autoclave Note: for motility assays, mage agar 0.22%
 * 1 L dH20
 * 6 g Bacto tryptone
 * 3 g Bacto yeast extract
 * 0.5 g CaCl2·2H20 (this is equal to 3.4 mM)
 * 12 g Bacto agar (optional)
 * 3 g agar + 250 mL broth per bottle

TY top agar (500 mL)
Autoclave Note: You do not need to add any additional calcium when performing phage transductions
 * 500 mL dH20
 * 3 g tryptone
 * 1.5 g yeast extract
 * 0.25 g CaCl2·2H20 (=3.4 mM)
 * 2 g agar

PH medium (1 L)
Autoclave
 * 1 L dH20
 * 4 g peptone
 * 0.5 g yeast extract
 * 0.5 g tryptone
 * 0.5 g CaCl2·2H20
 * 0.2 g MgSO4·7H20
 * 12 g agar (optional)
 * 3 g agar + 250 mL broth per bottle

PSY medium (1 L)
Autoclave NOTE: For culturing Bradyrhizobium japonicum, supplement with 0.1% arabinose
 * 1 L dH20
 * 20 g peptone
 * 20 g sucrose
 * 5 g yeast extract
 * 12 g agar (optional)
 * 3 g agar + 250 mL broth per bottle

YEM Broth (1 L)
Autoclave
 * 1 L dH20
 * 10 g mannitol
 * 1 g Bacto yeast extract
 * 500 mg K2HPO4
 * 200 mg MgSO4·7H20
 * 100 mg NaCl
 * 12 g agar (optional)
 * 3 g agar + 250 mL broth per bottle

Water agar (250 mL)
Autoclave
 * 250 mL dH20
 * * 3 g agar

Standard Minimal Medium (SMM) (250 mL)
To 250 mL sterile dH20 or water agar add: Autoclave Note: Very mucoid growth results from SMAJ at 1/2X&mdash;especially in sucrose
 * 2.5 mL 100X SMAJ
 * 2.5 mL 100X MCBT
 * 2.5 mL 30% carbon source
 * antibiotics (optional)

100X SMAJ (50 mL)
(takes a while to dissolve) QS to volume with ddH20 Filter sterilize Store at room temperature
 * ~30 mL ddH20
 * 8 g KH2PO4
 * 12 g K2HPO4
 * 2 g NaCl
 * 4 g NH4Cl

100X MCBT (50 mL)
Filter sterilize Note: Biotin takes a while to dissolve Store at room temperature
 * 50 mL ddH20
 * 2.46 g MgSO4·7H20
 * 1 mg CoCl2
 * 1 mg biotin
 * 3 mg thiamine

100X (30%) carbon source (50 mL)
Filter sterilize Store at room temperature Note: mannitol and lactose are soluble at 30% only upon heating
 * 15 g carbon source (e.g. sucrose, mannitol, glucose, lactose, sodium succinate).
 * QS to volume with ddH20

100X (2%) calcofluor (40 mL)
Filter sterilize Store at room temperature
 * 0.8 g calcofluor
 * QS to volume with ddH20

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
 * 1 L dH20
 * 0.35 g KH2PO4
 * 2 mL 500X magnesium sulfate
 * 2 mL 500X calcium chloride
 * 2 mL 500X minor salts
 * pH to 7.0 (~850 μL 2N KOH)
 * 13 g plant agar

Broth
Autoclave for 15 minutes Add:
 * 1 L dH20
 * 0.35 g KH2PO4
 * pH to 7.0 (~850 μL 2N KOH)
 * 1 mL 500X magnesium sulfate
 * 1 mL 500X calcium chloride
 * 1 mL 500X minor salts

500X magnesium sulfate (50 mL)
Filter sterilize
 * 50 mL ddH20
 * 6.25 g MgSO4·7H20

500X calcium chloride (50 mL)
Filter sterilize
 * 50 mL ddH20
 * 3.75 g CaCl2·2H20

500X minor salts (200 mL)
QS to volume with ddH20 Some heating may be required to get it all into solution Filter sterilize
 * 1.9 g Na2EDTA·2H20
 * 1.4 g FeSO4·7H20
 * 300 mg ZnSO4·7H20
 * 300 mg H3BO3
 * 300 mg MnSO4·H20
 * 30 mg Na2MoO4·2H20
 * 3 mg CuSO4
 * 3 mg CoCl2

500X Potassium nitrate (40 mL)
Filter sterilize
 * 40 mL ddH2O
 * 4.04 g potassium nitrate (KNO3)