Source: Protocol Online 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. Date Added: 2011-08-16 Date Modified: 2011-08-16 Procedure - 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 65°C for 1 hour.
- Purify with PCR cleanup kit (Qiagen)
- Spec with Nanodrop
- Run 200 ng at 90V for 1 hour on a 1% agarose gel with EtBr.
Note - 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.
- 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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