Griffitts:Common media: Difference between revisions
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* [[Griffitts:Stock solutions#Antibiotics|antibiotics]] (optional)<br> | * [[Griffitts:Stock solutions#Antibiotics|antibiotics]] (optional)<br> | ||
[[Wikipedia:Autoclave|Autoclave]]<br> | [[Wikipedia:Autoclave|Autoclave]]<br> | ||
Note: Very mucoid growth results from [[Griffitts:Common media#100X SMAJ (50 mL)|SMAJ]] at 1/2X | Note: Very mucoid growth results from [[Griffitts:Common media#100X SMAJ (50 mL)|SMAJ]] at 1/2X—especially in sucrose | ||
===100X SMAJ (50 mL)=== | ===100X SMAJ (50 mL)=== |
Revision as of 13:01, 17 January 2008
Bacterial Media and Additives
LB Broth (1 L)
- 1 L dH20
- 10 g Bacto tryptone
- 5 g Bacto yeast extract
- 5 g NaCl
- 1 mL 2N NaOH
- 12 g agar (optional)
Autoclave
Note: Add 3 mL 1 M CaCl2 before phage transductions
LB-Top agar (500 mL)
- 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
- Microwave until agar is dissolved
Note: Add 1.5 mL 1 M CaCl2 before phage transductions
Swarm agar for E. coli (400 mL)
- 400 mL dH20
- 4 g Tryptone
- 2 g NaCl
- 0.88 g Agar (for 0.22%)
Autoclave
TY broth (1 L)
- 1 L dH20
- 6 g Bacto tryptone
- 3 g Bacto yeast extract
- 0.5 g CaCl2·2H20
- 12 g Bacto agar (optional)
Autoclave
Note: for motility assays, mage agar 0.22%
TY top agar (500 mL)
- 500 mL dH20
- 3 g Bacto tryptone
- 1.5 g Bacto yeast extract
- 0.25 g CaCl2·2H20
- 2 g Bacto agar
Autoclave
Note: You do not need to add any additional calcium when performing phage transductions
Water agar (250 mL)
- 250 mL dH20
- * 3 g agar
Standard Minimal Medium (SMM)(250 mL)
To 250 mL sterile dH20 or water agar add:
- 2.5 mL 100X SMAJ
- 2.5 mL 100X MCBT
- 2.5 mL 30% carbon source
- antibiotics (optional)
Autoclave
Note: Very mucoid growth results from SMAJ at 1/2X—especially in sucrose
100X SMAJ (50 mL)
- ~30 mL ddH20
- 8 g KH2PO4
- 12 g K2HPO4
- 2 g NaCl
- 4 g NH4Cl
(takes a while to dissolve)
QS to volume with ddH20
Filter sterilize
Store at room temperature
100X MCBT (50 mL)
Filter sterilize
Note: Biotin takes a while to dissolve
Store at room temperature
100X (30%) carbon source (50 mL)
- 15 g carbon source (e.g. sucrose, mannitol, glucose, lactose, sodium succinate).
- QS to volume with ddH20
Filter sterilize
Store at room temperature
Note: mannitol and lactose are soluble at 30% only upon heating
Plant Media and Additives
Standard Nodulation Medium (SNM) (1 L)
Agar slants
- 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
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 dH20
- 0.35 g KH2PO4
- pH to 7.0 (~850 μL 2N KOH)
Autoclave for 15 minutes
Add:
- 2 mL 500X magnesium sulfate
- 2 mL 500X calcium chloride
- 2 mL 500X minor salts
500X magnesium sulfate (50 mL)
- 50 mL dH20
- 6.25 g MgS04·7H20
Filter sterilize
500X calcium chloride (50 mL)
- 50 mL dH20
- 3.75 g CaCl2·2H20
Filter sterilize
500X minor salts (200 mL)
- 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
QS to volume with ddH20
Some heating may be required to get it all into solution
Filter sterilize