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Solid Media Protocol

Solution A

Sterile filter solution with:

• 2 g of Na2S2O3
• 10 mL of H2O

Solution B

Autoclave solution of (2.5% tryptone):

• 2.5g tryptone broth
• 100 mL of H2O

Solution C

Sterile filter solution of:

• 500 mL deionized water
• 27 g (NH4)2SO4
• 0.9 g KCl
• 1.605 g leucine
• 4.5 g MgSO4
• 0.02 g HK2PO4
• 0.485 g diaminopimelic acid
• ~0.7 mL H2SO4 (or enough to bring pH down to ~2.0)
• 20 g Fe(II)SO4 (after adding everything else above)

Solution D

Autoclave solution containing (4% agarose):

• 20 g Agarose
• 500 mL distilled water

1. Heat up agarose solution until it becomes liquid
2. Pick a volume of solid media to make
3. Mix 1% vol A & 50% vol D, and 1% vol B & 48% vol C
4. Mix together AD and BC
5. Pour into petri dishes and allow to harden

Credit for protocol goes to Jason Candreva, Research Associate, Columbia University

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