Chromatin Immunoprecipitation: Difference between revisions
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=== Reverse Crosslinks of Protein/DNA Complexes to Free DNA=== | === 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. | 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. | ||
37. To all tubes, add 1 μL of RNase A and incubate for 30 minutes at 37°C. | 37. To all tubes, add 1 μL of RNase A and incubate for '''30 minutes''' at 37°C. | ||
38. 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 | 38. 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. | '''1-2 hours'''. | ||
==== Purification of ChIP DNA ==== | ==== Purification of ChIP DNA ==== | ||