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Revision as of 03:50, 4 October 2012 (view source) Iamannamai (Talk | contribs) (→Solid Media Protocol) ← Older edit		Revision as of 03:00, 27 October 2012 (view source) FourEyeGuy1962 (Talk | contribs) (→Solid Media Protocol) Newer edit →
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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>
Line 205:		Line 205:
	'''Solution C'''		'''Solution C'''
	<BLOCKQUOTE>		<BLOCKQUOTE>
-	Autoclave solution of:	+	Sterile filter solution of:
	<UL>		<UL>
	<LI>500 mL deionized water		<LI>500 mL deionized water
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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>10 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

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Revision as of 03:00, 27 October 2012

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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)
• 10 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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