Editing M9 (section)

== Reagents/Materials:  ==

Erlenmeyer flask of volume at 2X for amount of media to be made (i.e. 1L flask to make 500mL)<br> M9 minimal salts solution (5X concentrate)<br> 

*To 800mL of distilled/deionized water add: 
*64g sodium phosphate, penta-hydrate -- Na2HPO4-7H2O 
*15g potassium phosphate (dibasic) -- KH2PO4 
*2.5g table salt -- NaCl 
*5.0g ammonium chloride -- NH4Cl 
*Stir until dissolved. 
*Adjust volume to 1000mL (1L).

Sterilze by autoclaving or filter sterilization if autoclave is not available. <br> 

Distilled water 

<br> 1M solution of magnesium sulfate (MgSO4).<br> 

20% solution wt/wt of glucose (20g gulcose in 80g of water). Add powdered glucose (also called "dextrose") to water solution slowly while mixing, else the glucose will clump and not easily dissolve. Filter sterilize when finished - do not autoclave as the heat will carmelize the sugar.<br> 

1M solution of calcium chloride (CaCl2) 

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To make solid media, include agar base and sterile Petrie plates. 

=== Method  ===

To prepare 1L of media, add:<br> 

*200mL 5X M9 salts solution 
*to ~800mL of distilled water 
*Add 15g of agar media if agar plates are to be poured.

After autoclaving, swirl to mix evenly and "temper" at room temperature until you can place your hand on the flask for 2 second (an adult should do this!), then add the following: 

*2mL of 1M MgSO4 solution 
*0.1mL of 1M CaCl2 solution 
*20mL of 20% glucose

Swirl to mix evenly. 

Liquid media should be distributed to sterile bottles or tubes with a pipette and pipettor. Small bottles can hold 250mL of media for use when needed, for instance, or aliquots of 5mL or 10mL can be transferred to sterile, capped test tubes. 

If plates are to be poured, ~25mL of media should be added per Petrie dish and allowed to solidify at room temperature. Poured/solid plates can be stacked and replaced in the original plastic sleeve in which they came. Label the sleeve with the type of media and date poured. <br> 

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=== Protocol 2  ===

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1. In a 4 liter Erlenmeyer flask, combine: 

*12.8 grams Na<sub>2</sub>HPO4 
*3.0 grams KH<sub>2</sub>PO<sub>4</sub> 
*0.5 grams NaCl 
*1.0 gram NH4Cl

2. Bring to approximately 950 ml with ddH<sub>2</sub>O and pH to 7.4 with NaOH.<br> 

3. Autoclave for 20 minutes on slow exhaust. Store at 4oC when finished.<br> 

4. Dissolve the following in 5-10 ml of ddH<sub>2</sub>O in a test tube and cold filter, using a 10 ml sterile syringe and a 0.22 micron filter, into the autoclaved media:<br> 

*2.0 grams glucose 
*0.4940 grams MgSO<sub>4</sub>.7H<sub>2</sub>O 
*0.0152 grams CaCl<sub>2</sub>.2H<sub>2</sub>O 
*0.0100 grams Thiamine 
*0.0100 grams FeSO<sub>4</sub>.7H<sub>2</sub>O

<br>5. Or, dissolve each in a total volume of approximately 50 ml of ddH2O and cold filter that into the media.<br> 

6. After filtering each chemical into the media, rinse the test tube with ddH<sub>2</sub>O and filter the ddH<sub>2</sub>O into the media also. When finished, rinse the filter really well with ddH<sub>2</sub>O and bring to 1 liter.<br> 

7. Prior to inoculation, add the appropriate antiobiotic/s. 

=== <br>Protocol 3<br>  ===

1. Make M9 salts<br> To make 10X M9 Salts aliquot 800ml H2O and add<br> 

*60 g Na<sub>2</sub>HPO<sub>4</sub>.7H<sub>2</sub>O<br>
*30 g KH<sub>2</sub>PO<sub>4</sub><br>
*5 g NaCl<br>
*10 g NH<sub>4</sub>Cl<br>add water to 1000 ml and autoclave <br>

Stir until dissolved<br> Adjust to 1000ml with distilled H2O<br> Sterilize by autoclaving<br> 

2. Make medium 

Measure ~700ml of distilled H2O (sterile)<br> 

*Add 100ml of 10X M9 salts 
*Add 2ml of 1M MgSO<sub>4</sub> (sterile) 
*Add 20 ml of 20% glucose (or other carbon source) 
*Add 100 ul of 1M CaCl<sub>2</sub> (sterile)

Adjust to 1000 ml with distilled H2O<br>

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[[Category:Buffer_Recipe]]