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Rapid Chromatin De-crosslinking

Author: Scott Smemo
Source: Protocol Online
Date Added: Tue Aug 16 2011
Date Modified: Tue Aug 16 2011
Abstract: For checking how well one's chromatin is sonicated, this is a fast (2 hour total) alternative to standard methods and yields nearly identical results.

Procedure

  1. In a single tube, add:
    10 ul chromatin
    85 ul water
    4 ul NaCl (5M)
    8 ul Proteinase K (20 mg/ml)
    4 ul RNase (mg/ml)

    Incubate at 65C for 1 hour.
     
  2. Purify with PCR cleanup kit (Qiagen)
  3. Spec with Nanodrop
  4. Run 200 ng at 90V for 1 hour on a 1% agarose gel with EtBr.

Note

  1. In the standard protocols I have found, decrosslinking takes a minimum of 6 hours. For accurate input DNA quantitation, an RNase digest (at least 30 mins) is necessary. Furthermore, a Proteinase K digest is required, adding at least another hour. This adds up to making it unpleasant to do in a single day, so usually it gets done overnight and you lose a day. The above method will let you sonicate, check for size and re-sonicate or IP all in the same day. Run side-by-side, this method yields results nearly identical to the standard protocol in a fraction of the time.
  2. It may be important to say we crosslink in 1.8% formaldehyde, aim for 100-200 bp fragments, and in our trials a 10 ul chromatin sample yields 1-2 ug of DNA (We start with primary tissue, so I can't tell you how many cells that corresponds to). Starting with more material may affect the times required for digestion.
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