ChIP-Chip E. coli - Revision history

Torsten Waldminghaus: /* References */

Torsten Waldminghaus — Wed, 07 Jul 2010 10:37:00 GMT

References

←Older revision		Revision as of 10:37, 7 July 2010
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	==References==		==References==
		+
		+
		+	<biblio>
		+	#Waldminghaus-2010 pmid=20602746
		+	</biblio>

	*Glycogen does not interfere with Klenow random labeling http://www.fermentas.com/catalog/reagents/glycogen.htm		*Glycogen does not interfere with Klenow random labeling http://www.fermentas.com/catalog/reagents/glycogen.htm

Torsten Waldminghaus: /* Reagents */

Torsten Waldminghaus — Tue, 02 Mar 2010 08:10:30 GMT

Reagents

←Older revision		Revision as of 08:10, 2 March 2010
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	\|1 mM EDTA\|\|100µl 0.5M		\|1 mM EDTA\|\|100µl 0.5M
	\|-		\|-
-	\|0.5% Nonidet-P40\|\|250µl	+	\|0.5% Nonidet-P40 (=Triton X114)\|\|250µl
	\|-		\|-
	\|0.5% Sodium deoxycholate\|\|2.5ml 10%		\|0.5% Sodium deoxycholate\|\|2.5ml 10%

Torsten Waldminghaus: /* Procedure */

Torsten Waldminghaus — Mon, 01 Mar 2010 14:24:35 GMT

Procedure

←Older revision		Revision as of 14:24, 1 March 2010
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	*Add 80 μl TE and 1µl RNase A (20mg/ml) to the eluted DNA and to 100µl of the control DNA (see above)		*Add 80 μl TE and 1µl RNase A (20mg/ml) to the eluted DNA and to 100µl of the control DNA (see above)

-	*Incubate for 30 min at 42°C	+	*Incubate for 90 min at 42°C

	* add 20 μl Proteinase K to each tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl). (Note: many protocols no longer have this step; I get equivalent data when I skip this)		* add 20 μl Proteinase K to each tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl). (Note: many protocols no longer have this step; I get equivalent data when I skip this)

Torsten Waldminghaus: /* Reagents */

Torsten Waldminghaus — Mon, 01 Mar 2010 14:17:07 GMT

Reagents

←Older revision		Revision as of 14:17, 1 March 2010
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	\|+ '''Wash-Buffer'''		\|+ '''Wash-Buffer'''
	\|-		\|-
-	\|10mM Tris pH 8.0\|\|	+	\|10mM Tris pH 8.0\|\|500µl 1M
	\|-		\|-
-	\|250 mM LiCl\|\|	+	\|250 mM LiCl\|\|5 ml 2.5M
	\|-		\|-
-	\|1 mM EDTA\|\|	+	\|1 mM EDTA\|\|100µl 0.5M
	\|-		\|-
-	\|0.5% Nonidet-P40\|\|	+	\|0.5% Nonidet-P40\|\|250µl
	\|-		\|-
-	\|0.5% Sodium deoxycholate\|\|	+	\|0.5% Sodium deoxycholate\|\|2.5ml 10%
	\|-		\|-
	\|}		\|}

Torsten Waldminghaus: /* Reagents */

Torsten Waldminghaus — Tue, 09 Feb 2010 11:19:02 GMT

Reagents

←Older revision		Revision as of 11:19, 9 February 2010
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	\|+ '''IP-Buffer (Immunoprecipitation Buffer)'''		\|+ '''IP-Buffer (Immunoprecipitation Buffer)'''
	\|-		\|-
-	\|50 mM HEPES-KOH pH 7.5\|\|	+	\|50 mM HEPES-KOH pH 7.5\|\|50ml 0.5M
	\|-		\|-
-	\|150 mM NaCl\|\|	+	\|150 mM NaCl\|\|15ml 5M
	\|-		\|-
-	\|1 mM EDTA\|\|	+	\|1 mM EDTA\|\|1ml 0.5M
	\|-		\|-
-	\|1% Triton X 100\|\|	+	\|1% Triton X 100\|\|5ml
	\|-		\|-
-	\|0.1 % Sodium deoxycholate\|\|	+	\|0.1 % Sodium deoxycholate\|\|0.5g
	\|-		\|-
-	\|0.1 % SDS\|\|	+	\|0.1 % SDS\|\|5ml 10%
	\|-		\|-
	\|}		\|}

Torsten Waldminghaus: /* Reagents */

Torsten Waldminghaus — Tue, 18 Aug 2009 07:55:26 GMT

Reagents

←Older revision		Revision as of 07:55, 18 August 2009
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	{\| border="3"		{\| border="3"
-	!Final concentration !! For ~~500ml~~	+	!Final concentration !! For 50ml
	\|+ '''Wash-Buffer'''		\|+ '''Wash-Buffer'''
	\|-		\|-
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	{\| border="3"		{\| border="3"
-	!Final concentration !! For ~~500ml~~	+	!Final concentration !! For 10ml
	\|+ '''Elution-Buffer'''		\|+ '''Elution-Buffer'''
	\|-		\|-

Torsten Waldminghaus at 08:30, 20 April 2009

Torsten Waldminghaus — Mon, 20 Apr 2009 08:30:20 GMT

←Older revision		Revision as of 08:30, 20 April 2009
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-	*500 ml '''IP-Buffer (Immunoprecipitation Buffer)''':	+	{\| border="3"
-	**50 mM HEPES-KOH pH 7.5	+	!Final concentration !! For 500ml
-	**150 mM NaCl	+	\|+ '''IP-Buffer (Immunoprecipitation Buffer)'''
-	**1 mM EDTA	+	\|-
-	**1% Triton X 100	+	\|50 mM HEPES-KOH pH 7.5\|\|
-	**0.1 % Sodium deoxycholate	+	\|-
-	**0.1 % SDS	+	\|150 mM NaCl\|\|
		+	\|-
		+	\|1 mM EDTA\|\|
		+	\|-
		+	\|1% Triton X 100\|\|
		+	\|-
		+	\|0.1 % Sodium deoxycholate\|\|
		+	\|-
		+	\|0.1 % SDS\|\|
		+	\|-
		+	\|}

-	*30 ml IP-Buffer + 500 mM NaCl

-	*~~30 ml~~ '''Wash-Buffer''':	+	*IP-Buffer with 500 mM NaCl: add 2.8mL 5M NaCl to 40mL IP-buffer
-	**10mM Tris pH 8.0	+
-	**250 mM LiCl	+
-	**1 mM EDTA	+	{\| border="3"
-	**0.5% Nonidet-P40	+	!Final concentration !! For 500ml
-	**0.5% Sodium deoxycholate	+	\|+ '''Wash-Buffer'''
		+	\|-
		+	\|10mM Tris pH 8.0\|\|
		+	\|-
		+	\|250 mM LiCl\|\|
		+	\|-
		+	\|1 mM EDTA\|\|
		+	\|-
		+	\|0.5% Nonidet-P40\|\|
		+	\|-
		+	\|0.5% Sodium deoxycholate\|\|
		+	\|-
		+	\|}
		+
		+
		+	{\| border="3"
		+	!Final concentration !! For 500ml
		+	\|+ '''Elution-Buffer'''
		+	\|-
		+	\|50 mM Tris (pH 7.5)\|\|500μL 1M
		+	\|-
		+	\|10 mM EDTA\|\|200μL 0.5M
		+	\|-
		+	\|1% SDS\|\|1mL 10%
		+	\|-
		+	\|ddH<sub>2</sub>O\|\|8.3mL
		+	\|-
		+	\|}

-	*10 ml '''Elution-Buffer''':
-	**50 mM Tris (pH 7.5)
-	**10 mM EDTA
-	**1% SDS

	'''For labeling the DNA:'''		'''For labeling the DNA:'''

Torsten Waldminghaus at 08:10, 20 April 2009

Torsten Waldminghaus — Mon, 20 Apr 2009 08:10:18 GMT

←Older revision		Revision as of 08:10, 20 April 2009
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	*1 ml 100mM [[PMSF]] or 0.5 ml 250 mM [[Pefabloc]]		*1 ml 100mM [[PMSF]] or 0.5 ml 250 mM [[Pefabloc]]

-	*50 ml '''Lysis Buffer''':	+
-	**10mM Tris (pH 8.0)	+	{\| border="3"
-	**20% sucrose	+	!Final concentration !! For 50ml
-	**50mM NaCl	+	\|+ '''Lysis Buffer'''
-	**10mM EDTA	+	\|-
-	**10 mg/ml lysozyme	+	\|10mM Tris (pH 8.0)\|\|500μL 1M
		+	\|-
		+	\|20% sucrose\|\|10g
		+	\|-
		+	\|50mM NaCl\|\|500μL 5M
		+	\|-
		+	\|10mM EDTA\|\|1mL 0.5M
		+	\|-
		+	\|10 mg/ml lysozyme\|\|0.5g
		+	\|-
		+	\|}
		+	*Note: Prepare the 50ml without lysozyme and than add the corresponing ammount to 10 ml aliquotes and store them ad -20°C
		+

	*500 ml '''IP-Buffer (Immunoprecipitation Buffer)''':		*500 ml '''IP-Buffer (Immunoprecipitation Buffer)''':

Torsten Waldminghaus: /* Procedure */

Torsten Waldminghaus — Mon, 20 Apr 2009 07:40:04 GMT

Procedure

←Older revision		Revision as of 07:40, 20 April 2009
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	*Prepare the hybridisation buffer by pipetting the following into a tube. CARE Formamide is toxic.		*Prepare the hybridisation buffer by pipetting the following into a tube. CARE Formamide is toxic.
	{\| border="3"		{\| border="3"
-	! Component !! Volume for 125μL hybridisation (μL)	+	! Component !! Volume for 125μL hybridisation (μL)!! Volume for one slide with four arrays
	\|-		\|-
-	\|12xMES \|\|10	+	\|12xMES \|\|10 \|\|50
	\|-		\|-
-	\|5M Sodium chloride \|\|24	+	\|5M Sodium chloride \|\|24\|\|120
	\|-		\|-
-	\|Formamide \|\|24	+	\|Formamide \|\|24\|\|120
	\|-		\|-
-	\|0.5M EDTA \|\|5	+	\|0.5M EDTA \|\|5\|\|25
	\|-		\|-
-	\|10% Triton X100 \|\|12	+	\|10% Triton X100 \|\|12\|\|60
	\|}		\|}
-	*The given volumes are for one hybridisation. If you have more than one you should do a master mix and use 75μL in the following step.	+	*The given volumes in the first column are for one hybridisation. If you have more than one you should do a master mix (second column) and use 75μL in the following step.
	*In a different tube join the differentialy labeled test and control DNA as follows:		*In a different tube join the differentialy labeled test and control DNA as follows:
	{\| border="3"		{\| border="3"

Torsten Waldminghaus: /* Abstract */

Torsten Waldminghaus — Mon, 20 Apr 2009 07:36:34 GMT

Abstract

←Older revision		Revision as of 07:36, 20 April 2009
Line 8:		Line 8:

	==Abstract==		==Abstract==
-	ChIP-Chip stands for <u>Ch</u>romatin <u>I</u>mmuno<u>p</u>recipitation and chip in the sense of DNA microarray. It is a technique to determine the genome-wide binding sites of a DNA-binding protein. While the basic principle is the same for all species, there are some differences in handling cells. This protocol is developed and tested for '''E. coli'''. It should work the same way for other bacteria but that remains to be proven. Published protocols also exist for other bacterial species, including <i>Bacillus subtilis</i>, <i>Caulobacter crescentus</i> and <i>Mycobacterium tuberculosis</i>.	+	ChIP-Chip stands for <u>Ch</u>romatin <u>I</u>mmuno<u>p</u>recipitation and chip in the sense of DNA microarray. It is a technique to determine the genome-wide binding sites of a DNA-binding protein. While the basic principle is the same for all species, there are some differences in handling cells. This protocol is developed and tested for '''''E. coli'''''. It should work the same way for other bacteria but that remains to be proven. Published protocols also exist for other bacterial species, including <i>Bacillus subtilis</i>, <i>Caulobacter crescentus</i> and <i>Mycobacterium tuberculosis</i>.

	Because <i>E. coli</i> can be grown to high cell densities relative to eukaryotes, it is possible to generate sufficient DNA to label without using a PCR-based method. This method uses strand displacement primer extension with Klenow DNA polymerase and amplifies the DNA ~10-fold, while simultaneously incorporating dye-coupled nucleotide.		Because <i>E. coli</i> can be grown to high cell densities relative to eukaryotes, it is possible to generate sufficient DNA to label without using a PCR-based method. This method uses strand displacement primer extension with Klenow DNA polymerase and amplifies the DNA ~10-fold, while simultaneously incorporating dye-coupled nucleotide.