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→Reverse cross-linking and recover ChIP DNA
# Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the magnet to facilitate supernatant recovery.
# Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to complete the reversal of the formaldehyde cross-links.
==== Purification of ChIP DNA ====
# 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.
# 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.
# Wash the column with 750 µl Qiagen Buffer PE, centrifuge for 30-60sec @ 13,000 RPM, empty, and centrifuge the column containing the bound DNA a second time allow it to dry.
# 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).
===Analysis of Immunoprecipitated DNA===
* See [[RT-PCR primer design for ChIP]] to design primers if analysing by qPCR
* Dilute eluted DNA 250X