Waldminghaus:Back Door/Procedure ChIP-Chip - Revision history

Nadine Zimmer at 20:45, 29 November 2012

Nadine Zimmer — Thu, 29 Nov 2012 20:45:54 GMT

←Older revision		Revision as of 20:45, 29 November 2012
Line 20:		Line 20:
	[[Image:Pause point.png]] You can freeze the cell pellet and proceed later		[[Image:Pause point.png]] You can freeze the cell pellet and proceed later

-	*Resuspend in 300µl IP-Buffer	+	*Resuspend in 300µl IP-Buffer with 1mM Pefabloc

-	*Sonication in 1.5 ml tube 48 cycles, 30 sec. sonication, 30 sec. cooling	+	*Sonication in 1.5 ml tube 48 cycles, 30 sec. sonication, 30 sec. cooling with Bioruptor Plus from diagenode

-	*mix 2 x 150µl sonication sample with 1.5 ml IP Buffer each in a new tube	+	*mix 2 x 150µl sonication sample with 1.5 ml IP Buffer containing 1mM Pefabloc each in a new tube
-		+
-	*Add 16.5 μl Pefabloc of 100mM => final concentration of 10mM	+

	*Centrifuge 12,000 g, 4 °C, 10 min		*Centrifuge 12,000 g, 4 °C, 10 min
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	*Incubate for 90 min at 42°C		*Incubate for 90 min at 42°C

-	*add 15 μl Proteinase K to each tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl).	+	*add 15 μl Proteinase K ( Conc.:20 mg/ml ) to each tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl).

	*To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. If not using Proteinase K, incubate overnight at 65 °C, or boil samples for 10 minutes.		*To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. If not using Proteinase K, incubate overnight at 65 °C, or boil samples for 10 minutes.

Torsten Waldminghaus at 09:45, 20 November 2012

Torsten Waldminghaus — Tue, 20 Nov 2012 09:45:19 GMT

←Older revision		Revision as of 09:45, 20 November 2012
Line 24:		Line 24:
	*Sonication in 1.5 ml tube 48 cycles, 30 sec. sonication, 30 sec. cooling		*Sonication in 1.5 ml tube 48 cycles, 30 sec. sonication, 30 sec. cooling

-	*mix 150µl sonication sample with 1.5 ml IP Buffer in a new tube	+	*mix 2 x 150µl sonication sample with 1.5 ml IP Buffer each in a new tube

	*Add 16.5 μl Pefabloc of 100mM => final concentration of 10mM		*Add 16.5 μl Pefabloc of 100mM => final concentration of 10mM

Nadine Zimmer: New page: This protocol has been broken up into 3 "days" but for E. coli it is possible to perform the entire ChIP experiment in a single day as well as the amplification/labeling step (which...

Nadine Zimmer — Thu, 11 Oct 2012 15:24:56 GMT

New page: This protocol has been broken up into 3 "days" but for E. coli it is possible to perform the entire ChIP experiment in a single day as well as the amplification/labeling step (which...

New page

This protocol has been broken up into 3 "days" but for E. coli it is possible to perform the entire ChIP experiment in a single day as well as the amplification/labeling step (which can be done overnight).

''Formaldehyde cross link and sonication:''

*50ml culture in [[LB]] or [[AB medium]] at 30 or 37 °C until OD600 0.5

*Add 27μl formaldehyde (37%) per ml medium (substract what you took out for messuring OD) => final concentration of about 1%

*Shake slowly (100 RPM) for 20 min at RT

*Add 200µl glycine (2.5M) per ml medium => final concentration of about 0.5 M

*Keep shaking for 5 min

*Harvest 50 ml of cells for each DNA-preparation (centrifuge 2500 g, 4°C, 10 min)

*Wash twice in cold 10 ml TBS (20mM; see Material) pH7.5

[[Image:Pause point.png]] You can freeze the cell pellet and proceed later

*Resuspend in 300µl IP-Buffer

*Sonication in 1.5 ml tube 48 cycles, 30 sec. sonication, 30 sec. cooling

*mix 150µl sonication sample with 1.5 ml IP Buffer in a new tube

*Add 16.5 μl Pefabloc of 100mM => final concentration of 10mM

*Centrifuge 12,000 g, 4 °C, 10 min

''IP:''

*Use 800 μl aliquot for one immunoprecipitation experiment

*Add 20 μl of a 50% slurry of protein A sepharose or protein A/G beads (note that the beads should be selected to work with the antibody being used)

*Add specific antibody (for example 5 μl Anti-seqA)

*Incubate at 4 °C for 60 minutes on a slow rotator.

*Collect sephareose beads by centrifugation for 2 min at 3500 rpm

*Pipett off supernatant and save as '''control DNA'''

*All following wash steps should be on a rotator at room temperature for 3 min with 2 min centrifugation as above:

#Wash '''twice''' with 500 μl I-Buffer
#Wash with 500 μl I-Buffer with 500 mM NaCl
#Wash with 500 μl Wash-Buffer
#Wash with 500 μl TE

*Add 100 μl of elution buffer. Gently pipet up and down two or three times in order to dislodge beads from the filter. Incubate 10 min in a 65 °C water bath. A water bath is used instead of other heating apparatuses in order to improve heat transfer.

*Centrifuge beads 2 min at 3500 rpm, room temperature.Transfer supernatant into new 1.5 ml tube.

*Add 85 μl TE/100µl sample and 2µl RNase A (10mg/ml) to the eluted DNA.

*Incubate for 90 min at 42°C

*add 15 μl Proteinase K to each tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl).

*To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. If not using Proteinase K, incubate overnight at 65 °C, or boil samples for 10 minutes.

*Purify DNA by phenol extraction and ethanol precipitation or using a PCR purification kit (e.g. from Qiagen)

*Elute or resuspend in 21.5 water

*Measure DNA-content ideally at a NanoDrop (should be around 0.2 to 0.4 μg)

*Use 1 and 10 ng DNA as template for quantitative PCR with primers that are specific for a known binding site of your DNA-binding protein and one negative control