Changes

* [[Dilution Buffer]]*[[Low Salt Immune Complex Wash Buffer]] * [[High Salt Immune Complex Wash Buffer]] * [[LiCl Immune Complex Wash Buffer]] make fresh (cold)* [[TE: 10 mM Tris 7.5, 0.1 mM EDTA (cold)Buffer]]

15. Prepare enough Dilution Buffer containing protease inhibitors for the number of desired immunoprecipitations and store on ice. 16. Each IP requires the addition of 900 μL of Dilution Buffer and 4.5 μL of Protease Inhibitor Cocktail II. 

* Prepare one microfuge tube containing 100 μL of sheared crosslinked chromatin (Section B, step 5) for the number of desired immunoprecipitations and put on ice. If chromatin has been previously frozen, thaw on ice.

 20. Incubate for '''1 hour ''' at 4°C with rotation. 

 25. Incubate '''overnight ''' at 4°C with rotation.

 26. Add 60 μL of Protein G Agarose to each IP and incubate for '''1 hour ''' at 4°C with rotation.

 28. Wash the Protein G Agarose-antibody/chromatin complex by resuspending the beads in 1 mL each of the cold buffers in the order listed below and incubating for '''3-5 minutes ''' on a rotating platform followed by brief centrifugation (3000-5000 x g for 1 minute) and careful removal of the supernatant fraction:** [[Low Salt Immune Complex Wash Buffer ]] (Catalog # 20-154), '''one wash'''** [[High Salt Immune Complex Wash Buffer ]] (Catalog # 20-155), '''one wash'''** [[LiCl Immune Complex Wash Buffer ]] (Catalog # 20-156), '''3-5 washes'''** [[TE Buffer ]] (Catalog # 20-157), '''two washes''' ''Note: for TE washes use a pipette to carefully aspirate, the beads seem to come off of the magnet easily with this wash'' ==== Elution of Protein/DNA Complexes ====

• * Bring 1 M NaHCO3 to room temperature. A precipitate may be observed but will go into solution once room temperature is achieved. The 1 M NaHCO3 can be vortexed.• * Set water bath to 65°C for use in Section Elater.129. Make Elution Buffer for all IP tubes as well as all Input tubes (see Section C, step 7).• * For each tube, prepare 200 μL of elution buffer as follows: 10 μL 20% SDS, 20 μL 1 M NaHCO3 and 170 μL sterile, distilled water.2. * OR can make this way [[ChIP Elution Buffer]]* Alternatively, make a large volume to accommodate all tubes. For example, if there are 10 tubes mix together 105 μL 20% SDS, 210 μL 1M NaHCO3 and 1.785 mL sterile, distilled water.330. For Input tubes (see Section C, step 7), add 200 μL of Elution Buffer and set aside at room temperature until Section E.431. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently.532. Incubate at room temperature for '''15 minutes'''.633. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes.734. Repeat steps 4-6 and combine eluates (total volume = 200 μL).E. === Reverse Crosslinks of Protein/DNA Complexes to Free DNA===136. To all tubes (IPs and Inputs) add 8 μL 5 M NaCl and incubate at 65°C for 4-5 hours or '''overnight ''' to reverse the DNA – Protein crosslinks. After this step the sample can be stored at -20°C and the protocol continued the next day.237. To all tubes, add 1 μL of RNase A and incubate for '''30 minutes ''' at 37°C.338. Add 4 μL 0.5M EDTA, 8 μL 1M Tris-HCl and 1 μL Proteinase K to each tube and incubate at 45°C for'''1-2 hours'''.

# 39. Add 5 volumes Qiagen Buffer PB (QIAquick PCR Purification Kit) to one volume of ChIP’d DNA. Add pH detector (at a 1:250 dilution) to samples. Upon addition of Buffer PB, the sample should be yellow, indicating the correct pH. If the sample is not yellow, the pH should be adjusted with 3M sodium acetate as recommended by the manufacturer (Qiagen). One microliter at a time, mixing between each works fine.# 40. Add half (~600 µl) of the solution to a QIAquick PCR Purification column, centrifuge for 30-60 sec @ 13,000 RPM , and then repeat with other half to bind the ~1.2 ml sample on a Qiagen column.# 41. Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM.# 42. Empty the collection tube and centrifuge the column containing the bound DNA a second time to allow it to dry.# 43. Elute the DNA from the column with two 35 µl aliquots (note: this is how much you will need to run duplicates with 5 primers and may need to be adjusted based on your experiment) of warmed (~55°C) Qiagen Buffer EB, allow to sit on column for 1 minute, spin for 1 min @ 13,000 RPM, and repeat).