Waldminghaus:Back Door/Procedure ChIP-Chip

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

You can freeze the cell pellet and proceed later

• Resuspend in 300µl IP-Buffer with 1mM Pefabloc

• 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 containing 1mM Pefabloc each in a new tube

• 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:

1. Wash twice with 500 μl I-Buffer
2. Wash with 500 μl I-Buffer with 500 mM NaCl
3. Wash with 500 μl Wash-Buffer
4. 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 ( 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.

• 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