Silver: ChIP-chip - Revision history

Shane M Buker: /* Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody hello */

2007-12-20T17:12:44Z

Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody hello

←Older revision		Revision as of 17:12, 20 December 2007
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-	==Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody ~~hello~~==	+	==Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody==
	===Crosslinking===		===Crosslinking===
	#Pick 5 colonies of each yeast strain and grow up in 10 ml YEPD overnight		#Pick 5 colonies of each yeast strain and grow up in 10 ml YEPD overnight

Shane M Buker: /* Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody */

2007-12-20T17:12:30Z

Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody

←Older revision		Revision as of 17:12, 20 December 2007
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-	==Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody==	+	==Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody hello==
	===Crosslinking===		===Crosslinking===
	#Pick 5 colonies of each yeast strain and grow up in 10 ml YEPD overnight		#Pick 5 colonies of each yeast strain and grow up in 10 ml YEPD overnight

ChrisBrown at 21:10, 21 September 2005

2005-09-21T21:10:30Z

←Older revision		Revision as of 21:10, 21 September 2005
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	==Day 8 - Klenow-Mediated Incorporation of Dyes/DNA Precipitation==		==Day 8 - Klenow-Mediated Incorporation of Dyes/DNA Precipitation==
		+	===Klenow-Mediated Incorporation of Dyes===
	*Cy5-dUTP = IP = blue		*Cy5-dUTP = IP = blue
	*Cy3-dUTP = WCE = pink		*Cy3-dUTP = WCE = pink

ChrisBrown at 21:09, 21 September 2005

2005-09-21T21:09:40Z

←Older revision		Revision as of 21:09, 21 September 2005
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-	==Day 1 - Crosslinking/Pre-Incubation of Beads with Antibody==	+	==Day 1-2 - Crosslinking/Pre-Incubation of Beads with Antibody==
	===Crosslinking===		===Crosslinking===
	#Pick 5 colonies of each yeast strain and grow up in 10 ml YEPD overnight		#Pick 5 colonies of each yeast strain and grow up in 10 ml YEPD overnight

ChrisBrown at 21:09, 21 September 2005

2005-09-21T21:09:04Z

←Older revision		Revision as of 21:09, 21 September 2005
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	5 30 sec 55°C		5 30 sec 55°C
	6 1 min 72°C		6 1 min 72°C
-	7 go to step 4 for 24x	+	7 go to step 4 for 24x
-	8 4 min 72°C	+	8 4 min 72°C
	9 infinity 10°C		9 infinity 10°C
		+	#After PCR amplification, check product on gel: 5 µl DNA, 5 µl H<sub>2</sub>O, 2 µl 6x DNA dye, no Bromphenol Blue (runs at same level as sonicated DNA), run with 1 kb and 100 bp ladders (1.2% agarose gel). Run for 30 min at 100V, look for a smear around 350-400bp
		+	#Use Qiagen PCR purification kit and elute in 65 µl of their elution buffer

-	8~~. After PCR amplification, check product on gel: 5 µl DNA, 5 µl H<sub>2</sub>O, 2 µl 6x DNA dye, no~~	+	==Day 8 - Klenow-Mediated Incorporation of Dyes/DNA Precipitation==
-	~~Bromphenol Blue (runs at same level as sonicated DNA), run with 1 kb and 100 bp~~	+	*Cy5-dUTP = IP = blue
-	~~ladders (1.2% agarose gel). Run for 30 min at 100V, look for a smear around 350~~-~~400bp~~	+	*Cy3-dUTP = WCE = pink
-	~~9. Use Qiagen PCR purification kit and elute in 65 µl of their elution buffer~~	+	#Transfer 21 µl of DNA to an eppendorf tube
-		+	#Add 20 µl of 2.5 x random primer solution (from BioPrime kit) to tube
-		+	#Boil for 5 min at 100°C, then put on ice, incubate for 5 min
-		+	#Add 5 µl of 10x dNTP (low T mix)
-		+	#*Low T dNTP Mix
-	~~BioPrime~~ Incorporation of Dyes	+	#**1.2 µl dATP
-		+	#**1.2 µl dCTP
-	Cy5-dUTP = IP = blue	+	#**1.2 µl dGTP
-	Cy3-dUTP = WCE = pink	+	#**0.6 µl dTTP
-		+	#**95.8 µl ddH<sub>2</sub>0
-	1. Transfer 21 µl of DNA to an eppendorf tube	+	#Add 3 µl of Cy5-dUTP to IP sample, and 3 µl of Cy3-dUTP to WCE sample, do in the dark since the dyes are light sensitive
-	2. Add 20 µl of 2.5 x random primer solution (from BioPrime kit) to tube	+	#Add 1 µl of high concentration Klenow (from BioPrime kit), make sure to mix well
-	3. Boil for 5 min at 100°C, then put on ice, incubate for 5 min	+	#Incubate at 37°C for 1 h (wrap in aluminum foil to keep dark)
-	4. Add 5 µl of 10x dNTP (low T mix)	+	#Use Qiagen purification kit to purify the DNA, elute in some considerable amount of EB buffer from kit (100 µl is fine-just want to get as much off the column as possible and the volume change doesn't matter since you'll be precipitating anyway)
-	~~BioPrime~~ dNTP Mix ~~(make your own-don't use theirs)~~	+	===DNA Precipitation===
-		+	#Add 12 µl of 3M NaOAc, 260 µl of 100% EtOH. Incubate at -20°C for at least a couple of days (wrap in aluminum foil to keep dark)
-	1.2 µl dATP	+	#Spin at 14k rpm, 4°C, 40 min
-	1.2 µl dCTP	+	#Wash with 500 µl 75% EtOH
-	1.2 µl dGTP	+	#Spin at 14k rpm, 4°C, 20 min
-	0.6 µl dTTP	+	#Air dry for at least 2 h. Should have nice, tight, bright pellets
-	95.8 µl ddH<sub>2</sub>0	+
-		+
-	5. Add 3 µl of Cy5-dUTP (to IP sample), and 3 µl of Cy3-dUTP to WCE sample~~. (~~do in the	+
-	dark~~---~~dyes are light sensitive)	+
-	6. Add 1 µl of high concentration Klenow (from BioPrime kit), make sure to mix well	+
-	7. Incubate at 37°C for 1 h (wrap in aluminum foil-keep dark)	+
-	8. Use Qiagen purification kit to purify the DNA, elute in some considerable amount of EB	+
-	buffer from kit (100 µl is fine-just want to get as much off the column as possible and the	+
-	volume change doesn't matter since you'll be precipitating anyway)	+
-	9. Add 12 µl of 3M NaOAc, 260 µl of 100% EtOH. Incubate at -20°C for at least a couple of	+
-	days (wrap ~~rack~~ in aluminum foil)	+
-	~~Pellet the DNA:~~	+
-	~~10.~~ Spin at 14k rpm, 4°C, 40 min	+
-	~~11.~~ Wash with 500 µl 75% EtOH	+
-	~~12.~~ Spin at 14k rpm, 4°C, 20 min	+
-	~~13.~~ Air ~~Dry~~. Should have nice, tight, bright pellets	+
-	~~14. Proceed to Microarray Hybridization~~	+

ChrisBrown at 21:02, 21 September 2005

2005-09-21T21:02:13Z

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ChrisBrown at 20:39, 21 September 2005

2005-09-21T20:39:10Z

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ChrisBrown at 19:55, 21 September 2005

2005-09-21T19:55:09Z

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ChrisBrown at 20:27, 20 September 2005

2005-09-20T20:27:05Z

←Older revision		Revision as of 20:27, 20 September 2005
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-	[[Silver: Protocols \| Protocols]]	+	Back to [[Silver Lab]]
		+
		+	Back to [[Silver: Protocols \| Protocols]]

ChrisBrown at 22:57, 19 September 2005

2005-09-19T22:57:46Z

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