Chromatin Immunoprecipitation: Difference between revisions
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* 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. | * 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. | ||
* 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. | * 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. | ||
30. For Input tubes (see Section C, step 7), add 200 μL of Elution Buffer and set aside at room temperature | |||
30. For Input tubes (see Section C, step 7), add 200 μL of Elution Buffer and set aside at room temperature. | |||
31. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently. | 31. Add 100 μL of Elution Buffer to each tube containing the antibody/agarose complex. Mix by flicking tube gently. | ||
32. Incubate at room temperature for 15 minutes. | 32. Incubate at room temperature for 15 minutes. | ||
33. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes. | 33. Pellet agarose by brief centrifugation (3000-5000 x g for 1 minute) and collect supernatant into new microfuge tubes. | ||
34. Repeat steps 4-6 and combine eluates (total volume = 200 μL). | 34. Repeat steps 4-6 and combine eluates (total volume = 200 μL). | ||