Team:Columbia-Cooper-NYC/Making Solid Media

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(→Solid Media Protocol)
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'''Solution B'''
'''Solution B'''
<BLOCKQUOTE>
<BLOCKQUOTE>
-
Adjust pH to 2.0 with sulfuric acid (H2SO4) and sterile filter solution with:
+
Autoclave solution of (2.5% tryptone):
<UL>
<UL>
-
<LI>2 g Fe(II)SO4
+
<LI>2.5g tryptone broth
-
<LI>10 mL of H2O
+
<LI>100 mL of H2O
</UL>
</UL>
</BLOCKQUOTE>
</BLOCKQUOTE>
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'''Solution C'''
'''Solution C'''
<BLOCKQUOTE>
<BLOCKQUOTE>
-
Autoclave solution of:
+
Sterile filter solution of:
-
<UL>500 mL deionized water
+
<UL>
 +
<LI>500 mL deionized water
<LI>27 g (NH4)2SO4
<LI>27 g (NH4)2SO4
<LI>0.9 g KCl
<LI>0.9 g KCl
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<LI>0.02 g HK2PO4
<LI>0.02 g HK2PO4
<LI>0.485 g diaminopimelic acid
<LI>0.485 g diaminopimelic acid
-
<LI>10 g Fe(II)SO4 (at the end)
+
<LI> ~0.7 mL H2SO4 (or enough to bring pH down to ~2.0)
 +
<LI>20 g Fe(II)SO4 (after adding everything else above)
</UL>
</UL>
</BLOCKQUOTE>
</BLOCKQUOTE>
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'''Solution D'''
'''Solution D'''
<BLOCKQUOTE>
<BLOCKQUOTE>
-
Autoclave solution containing:
+
Autoclave solution containing (4% agarose):
<UL>
<UL>
-
<LI>6 g Agarose
+
<LI>20 g Agarose
-
<LI>480 mL distilled water
+
<LI>500 mL distilled water
</UL>
</UL>
</BLOCKQUOTE>
</BLOCKQUOTE>
<OL>
<OL>
-
<LI>Mix A & D, and B & C
+
<LI>Heat up agarose solution until it becomes liquid
 +
<LI>Pick a volume of solid media to make
 +
<LI>Mix 1% vol A & 50% vol D, and 1% vol B & 48% vol C
<LI>Mix together AD and BC
<LI>Mix together AD and BC
<LI>Pour into petri dishes and allow to harden
<LI>Pour into petri dishes and allow to harden

Latest revision as of 03:01, 27 October 2012


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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