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In phase four, we will reverse our initial cross-linking proteins to DNA, followed by
purification of the DNA. Start by taking each IP sample as well as the input sample which
did not go through the preceding IP steps and add 8 microliters of 5 molar sodium
chloride to the 200 microliter samples followed by vortexing.
Incubate at 95 degrees Celsius for 15 minutes. To purify each sample we will use a DNA binding silica columns according
to the manufacturer's instructions. First add each sample for the proper amounts
of DNA binding buffer in vortex, then transfer each sample to a column placed inside a
collection tube. Centrifuge the columns at 15,000g for 1 minute.
Discard the flow through from the collection tube and add DNA wash buffer to each column.
Centrifuge the columns at 15000g for one minute. Discard the flow through from the collection tube
and spin the empty columns at 15000g for two minutes to ensure sure that they are completely dry.
Discard the old collection tube and place the purification column into a new clean tube.
Add 50 microliters of DNA elusion buffer directly to the membrane in the column.
Allow to sit for one minute then perform a final spin at 15000g for 1 minute.
Discard the purification column and save the purified DNA at -20 degrees Celsius until ready for the last phase.