29. 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.
30. * 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.3130. For Input tubes (see Section C, step 7), add 200 μL of Elution Buffer and set aside at room temperature until Section E.3231. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently.3332. Incubate at room temperature for 15 minutes.3433. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes.3534. Repeat steps 4-6 and combine eluates (total volume = 200 μL).
=== Reverse Crosslinks of Protein/DNA Complexes to Free DNA===
36. 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.