Griffitts:Common media: Difference between revisions
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==Bacterial Media and Additives== | ==Bacterial Media and Additives== | ||
=== | ===LB Broth (1 L)=== | ||
* 1 L dH<sub>2</sub> | * 1 L dH<sub>2</sub>O | ||
* 10 g Bacto [[Wikipedia:tryptone|tryptone]] | * 10 g Bacto [[Wikipedia:tryptone|tryptone]] | ||
* 5 g Bacto [[Wikipedia:yeast extract|yeast extract]] | * 5 g Bacto [[Wikipedia:yeast extract|yeast extract]] | ||
* 5 g NaCl | * 5 g NaCl | ||
* 1 mL | * 1 mL 2N NaOH | ||
* 12 g [[Wikipedia:Agar|agar]] (optional)<br> | * 12 g [[Wikipedia:Agar|agar]] (optional)<br> | ||
[[Wikipedia:Autoclave|Autoclave]] | ** 3 g agar + 250 mL broth per bottle | ||
[[Wikipedia:Autoclave|Autoclave]]<br> | |||
* 500 mL dH<sub>2</sub> | Note: Add 3 mL [[Griffitts:Stock solutions#1 M Calcium Chloride (75 mL)|1 M CaCl<sub>2</sub>]] before [[Griffitts:Transduction|phage transductions]] | ||
===LB-Top agar (500 mL)=== | |||
* 500 mL dH<sub>2</sub>O | |||
* 5 g Bacto [[Wikipedia:tryptone|tryptone]] | * 5 g Bacto [[Wikipedia:tryptone|tryptone]] | ||
* 2.5 g Bacto [[Wikipedia:yeast extract|yeast extract]] | * 2.5 g Bacto [[Wikipedia:yeast extract|yeast extract]] | ||
* 2.5 g NaCl | * 2.5 g NaCl | ||
* 0.5 mL | * 0.5 mL 2N NaOH | ||
* 2 g [[Wikipedia:Agar|agar]] | * 2 g [[Wikipedia:Agar|agar]]<br> | ||
[[Wikipedia:Autoclave|Autoclave]] | ** [[Wikipedia:Autoclave|Autoclave]]<br> | ||
Add | Note: Add 1.5 mL [[Griffitts:Stock solutions#1 M Calcium Chloride (75 mL)|1 M CaCl<sub>2</sub>]] before [[Griffitts:Transduction|phage transductions]] | ||
===Swarm agar for ''E. coli'' (400 mL)=== | |||
* 400 mL dH<sub>2</sub>O | |||
* 4 g Tryptone | |||
* 2 g NaCl | |||
* 0.88 g Agar (for 0.22%)<br> | |||
Autoclave | |||
===TY broth (1 L)=== | ===TY broth (1 L)=== | ||
* 1 L dH<sub>2</sub> | * 1 L dH<sub>2</sub>O | ||
* 6 g Bacto [[Wikipedia:tryptone|tryptone]] | * 6 g Bacto [[Wikipedia:tryptone|tryptone]] | ||
* 3 g Bacto [[Wikipedia:yeast extract|yeast extract]] | * 3 g Bacto [[Wikipedia:yeast extract|yeast extract]] | ||
* 0.5 g CaCl<sub>2</sub>·2H<sub>2</sub> | * 0.5 g CaCl<sub>2</sub>·2H<sub>2</sub>O (this is equal to 3.4 mM) | ||
* 12 g Bacto [[Wikipedia:Agar|agar]] (optional)<br> | * 12 g Bacto [[Wikipedia:Agar|agar]] (optional) | ||
[[Wikipedia:Autoclave|Autoclave]] | ** 3 g agar + 250 mL broth per bottle<br> | ||
Note: for [[Wikipedia:motility|motility]] assays, | [[Wikipedia:Autoclave|Autoclave]]<br> | ||
=== | Note: for [[Wikipedia:motility|motility]] assays, make agar 0.22% | ||
* 2.5 mL | ===TY top agar (500 mL)=== | ||
* 2 | * 500 mL dH<sub>2</sub>O | ||
* 2 | * 3 g tryptone | ||
* [[Wikipedia: | * 1.5 g yeast extract | ||
* 0.25 g CaCl<sub>2</sub>·2H<sub>2</sub>O (=3.4 mM) | |||
* 2 g agar<br> | |||
Autoclave<br> | |||
Note: You do not need to add any additional calcium when performing [[Griffitts:Transduction|phage transductions]] | |||
===PH medium (1 L)=== | |||
* 1 L dH<sub>2</sub>O | |||
* 4 g peptone | |||
* 0.5 g yeast extract | |||
* 0.5 g tryptone | |||
* 0.5 g CaCl<sub>2</sub>·2H<sub>2</sub>O | |||
* 0.2 g MgSO<sub>4</sub>·7H<sub>2</sub>O | |||
* 12 g agar (optional) | |||
** 3 g agar + 250 mL broth per bottle<br> | |||
Autoclave | |||
===PSY medium (1 L)=== | |||
* 1 L dH<sub>2</sub>O | |||
* 20 g peptone | |||
* 20 g sucrose | |||
* 5 g yeast extract | |||
* 12 g agar (optional) | |||
** 3 g agar + 250 mL broth per bottle<br> | |||
Autoclave<br> | |||
NOTE: For culturing ''Bradyrhizobium japonicum'', supplement with 0.1% arabinose | |||
===YEM Broth (1 L)=== | |||
* 1 L dH<sub>2</sub>O | |||
* 10 g mannitol | |||
* 1 g Bacto [[Wikipedia:yeast extract|yeast extract]] | |||
* 500 mg K<sub>2</sub>HPO<sub>4</sub> | |||
* 200 mg MgSO<sub>4</sub>·7H<sub>2</sub>O | |||
* 100 mg NaCl | |||
* 12 g [[Wikipedia:Agar|agar]] (optional)<br> | |||
** 3 g agar + 250 mL broth per bottle | |||
[[Wikipedia:Autoclave|Autoclave]]<br> | |||
===Water [[Wikipedia:agar|agar]] (250 mL)=== | |||
* 250 mL dH<sub>2</sub>O | |||
* * 3 g [[Wikipedia:Agar|agar]]<br> | |||
[[Wikipedia:Autoclave|Autoclave]] | [[Wikipedia:Autoclave|Autoclave]] | ||
===Standard Minimal Medium (SMM) (250 mL)=== | |||
To 250 mL sterile dH<sub>2</sub>O or [[Griffitts:Common media#Water agar (250 mL)|water agar]] add: | |||
* 2.5 mL [[Griffitts:Common media#100X SMAJ (50 mL)|100X SMAJ]] | |||
* 2.5 mL [[Griffitts:Common media#100X MCBT (50 mL)|100X MCBT]] | |||
* 2.5 mL [[Griffitts:Common media#100X 30% carbon_source (50 mL)|30% carbon source]] | |||
* [[Griffitts:Stock solutions#Antibiotics|antibiotics]] (optional)<br> | |||
Autoclave<br> | |||
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)=== | ||
* ~30 mL ddH<sub>2</sub> | * ~30 mL ddH<sub>2</sub>O | ||
* 8 g KH<sub>2</sub>PO<sub>4</sub> | * 8 g KH<sub>2</sub>PO<sub>4</sub> | ||
* 12 g K<sub>2</sub>HPO<sub>4</sub> | * 12 g K<sub>2</sub>HPO<sub>4</sub> | ||
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* 4 g NH<sub>4</sub>Cl<br> | * 4 g NH<sub>4</sub>Cl<br> | ||
(takes a while to dissolve)<br> | (takes a while to dissolve)<br> | ||
Filter sterilize | QS to volume with ddH<sub>2</sub>O<br> | ||
Filter sterilize<br> | |||
Store at room temperature | |||
===100X MCBT (50 mL)=== | ===100X MCBT (50 mL)=== | ||
* 50 mL ddH<sub>2</sub> | * 50 mL ddH<sub>2</sub>O | ||
* 2.46 g MgSO<sub>4</sub>·7H<sub>2</sub> | * 2.46 g MgSO<sub>4</sub>·7H<sub>2</sub>O | ||
* 1 mg CoCl<sub>2</sub> | * 1 mg CoCl<sub>2</sub> | ||
* 1 mg [[Wikipedia:biotin|biotin]] | * 1 mg [[Wikipedia:biotin|biotin]] | ||
* 3 mg [[Wikipedia:thiamine|thiamine]]<br> | * 3 mg [[Wikipedia:thiamine|thiamine]]<br> | ||
Filter sterilize | Filter sterilize<br> | ||
Note: Biotin takes a while to dissolve | Note: Biotin takes a while to dissolve<br> | ||
Store at room temperature | |||
===100X (30%) carbon source (50 mL)=== | ===100X (30%) carbon source (50 mL)=== | ||
* 15 g carbon source (e.g. [[wikipedia:sucrose|sucrose]], [[wikipedia:mannitol|mannitol]], [[wikipedia:glucose|glucose]], [[wikipedia:lactose|lactose]], [[wikipedia:succinate|sodium succinate]]). | * 15 g carbon source (e.g. [[wikipedia:sucrose|sucrose]], [[wikipedia:mannitol|mannitol]], [[wikipedia:glucose|glucose]], [[wikipedia:lactose|lactose]], [[wikipedia:succinate|sodium succinate]]). | ||
* QS to volume with ddH<sub>2</sub> | * QS to volume with ddH<sub>2</sub>O | ||
Filter sterilize | Filter sterilize<br> | ||
Store at room temperature<br> | |||
Note: mannitol and lactose are soluble at 30% only upon heating | Note: mannitol and lactose are soluble at 30% only upon heating | ||
==Plant Media== | ===100X (2%) calcofluor (40 mL)=== | ||
* 0.8 g calcofluor | |||
* QS to volume with ddH<sub>2</sub>O | |||
Filter sterilize<br> | |||
Store at room temperature<br> | |||
==Plant Media and Additives== | |||
===Standard Nodulation Medium (SNM) (1 L)=== | ===Standard Nodulation Medium (SNM) (1 L)=== | ||
* 1 L dH<sub>2</sub> | ====Agar slants==== | ||
* 1 L dH<sub>2</sub>O | |||
* 0.35 g KH<sub>2</sub>PO<sub>4</sub> | * 0.35 g KH<sub>2</sub>PO<sub>4</sub> | ||
* 2 mL 500X minor salts ( | * 2 mL [[Griffitts:Common media#500X magnesium sulfate (50 mL)|500X magnesium sulfate]] | ||
* pH to 7.0 (~850 μL 2[[Wikipedia:Concentration#Normality|N]] KOH | * 2 mL [[Griffitts:Common media#500X calcium chloride (50 mL)|500X calcium chloride]] | ||
* 2 mL [[Griffitts:Common media#500X minor salts (200 mL)|500X minor salts]] | |||
* pH to 7.0 (~850 μL 2[[Wikipedia:Concentration#Normality|N]] KOH) | |||
* 13 g plant [[Wikipedia:agar|agar]]<br> | * 13 g plant [[Wikipedia:agar|agar]]<br> | ||
Microwave to dissolve agar<br> | Microwave to dissolve agar<br> | ||
Distribute 8.2 mL per 18×150 tube<br> | Distribute 8.2 mL per 18×150 disposable tube<br> | ||
Cap tubes<br> | Cap tubes<br> | ||
[[Wikipedia:Autoclave|Autoclave]]<br> | [[Wikipedia:Autoclave|Autoclave]] for 10 minutes<br> | ||
Slant the racks and | 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 dH<sub>2</sub>O | |||
* 0.35 g KH<sub>2</sub>PO<sub>4</sub> | |||
* pH to 7.0 (~850 μL 2[[Wikipedia:Concentration#Normality|N]] KOH)<br> | |||
[[Wikipedia:Autoclave|Autoclave]] for 15 minutes<br> | |||
Add:<br> | |||
* 1 mL [[Griffitts:Common media#500X magnesium sulfate (50 mL)|500X magnesium sulfate]] | |||
* 1 mL [[Griffitts:Common media#500X calcium chloride (50 mL)|500X calcium chloride]] | |||
* 1 mL [[Griffitts:Common media#500X minor salts (200 mL)|500X minor salts]] | |||
===500X magnesium sulfate (50 mL)=== | |||
* 50 mL ddH<sub>2</sub>O<br> | |||
* 6.25 g MgSO<sub>4</sub>·7H<sub>2</sub>O<br> | |||
Filter sterilize | |||
===500X calcium chloride (50 mL)=== | |||
* 50 mL ddH<sub>2</sub>O<br> | |||
* 3.75 g CaCl<sub>2</sub>·2H<sub>2</sub>O<br> | |||
Filter sterilize | |||
===500X minor salts (200 mL)=== | ===500X minor salts (200 mL)=== | ||
* 1.9 g Na<sub>2</sub>[[Wikipedia:EDTA|EDTA]]·2H<sub>2</sub> | * 1.9 g Na<sub>2</sub>[[Wikipedia:EDTA|EDTA]]·2H<sub>2</sub>O | ||
* 1.4 g FeSO<sub>4</sub>·7H<sub>2</sub> | * 1.4 g FeSO<sub>4</sub>·7H<sub>2</sub>O | ||
* 300 mg ZnSO<sub>4</sub>·7H<sub>2</sub> | * 300 mg ZnSO<sub>4</sub>·7H<sub>2</sub>O | ||
* 300 mg H<sub>3</sub>BO<sub>3</sub> | * 300 mg H<sub>3</sub>BO<sub>3</sub> | ||
* 300 mg MnSO<sub>4</sub>·H<sub>2</sub> | * 300 mg MnSO<sub>4</sub>·H<sub>2</sub>O | ||
* 30 mg Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub> | * 30 mg Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O | ||
* 3 mg CuSO<sub>4</sub> | * 3 mg CuSO<sub>4</sub> | ||
* 3 mg CoCl<sub>2</sub> | * 3 mg CoCl<sub>2</sub> | ||
QS to volume with ddH<sub>2</sub> | QS to volume with ddH<sub>2</sub>O<br> | ||
Some heating may be required to get it all into solution<br> | |||
Filter sterilize | |||
===500X Potassium nitrate (40 mL)=== | |||
* 40 mL ddH<sub>2</sub>O | |||
* 4.04 g potassium nitrate (KNO<sub>3</sub>) | |||
Filter sterilize | Filter sterilize |
Latest revision as of 08:54, 16 August 2013
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 CaCl2 before phage transductions
LB-Top agar (500 mL)
- 500 mL dH2O
- 5 g Bacto tryptone
- 2.5 g Bacto yeast extract
- 2.5 g NaCl
- 0.5 mL 2N NaOH
- 2 g agar
Note: Add 1.5 mL 1 M CaCl2 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
- 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
- 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
- 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
Water agar (250 mL)
- 250 mL dH2O
- * 3 g agar
Standard Minimal Medium (SMM) (250 mL)
To 250 mL sterile dH2O 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 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)
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 ddH2O
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
- 1 L dH2O
- 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 dH2O
- 0.35 g KH2PO4
- pH to 7.0 (~850 μL 2N KOH)
Autoclave for 15 minutes
Add:
- 1 mL 500X magnesium sulfate
- 1 mL 500X calcium chloride
- 1 mL 500X minor salts
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 Na2EDTA·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