Biology Reference
In-Depth Information
4
Notes
1. The fi rst important parameter for ChIP is the quality of the
cross-linked DNA which needs to be effectively sonicated in
order to assure the relevance of the results with specifi c antibod-
ies and the target DNA segment of interest. Optimal DNA frag-
ment sizes for ChIP are 200-1,000 bp. DNA sonication
conditions must be determined empirically for each cell type and
sonicator model. Here are some general tips to ensure effi cient
sonication. First, use a sonicator with a microtip and perform
sonication in a 1.5 mL tube. Avoid sample foaming during soni-
cation, since this will decrease the effi ciency of chromatin shear-
ing. To avoid foaming, let the microtip of the sonicator reach the
bottom of 1.5 mL microfuge tube. To avoid excessive heating of
the sample, immerse the sample into an ice-water bath during
sonication, and keep at least 2 min intervals between each round
of sonication. Also, sonication using a series of short pulses is
more effi cient than a single long pulse. The numbers of pulse
sets should be optimized by prior experiments.
2. The quality of the antibody used for the chromatin immuno-
precipitation reactions is one of the most critical parameters of
ChIP. Antibodies of high specifi city and affi nity are required
for optimal results. Preimmune serum should be used to verify
specifi city with polyclonal antibodies and isotype-matched
nonspecifi c monoclonal antibody should be used to verify
specifi city with monoclonal antibodies.
3. The ChIP wash conditions are fully dependent on the proper-
ties of the antibodies chosen. This should be optimized prior
to the experiment. Our experience suggests that the wash con-
ditions provided in this protocol are suitable for most antibod-
ies tested. However, for certain antibodies, it might require
more a extensive washing regimen during the immunoprecipi-
tations to reduce background and discriminate positive con-
trols from negative controls. For example, fi ve times wash with
RIPA buffer (50 mM Tris-HCl, pH 8.0, 750 mM NaCl,
5 mM EDTA, 0.1 % SDS, 1 % Triton X-100, 0.1 % sodium
deoxycholic acid) is required for our IVa2 antibody.
4. Another important parameter for qPCR is that primer pairs
contain matching annealing temperatures (generally 55-60 °C)
and a high degree of sequence specifi city (20 bp for Ad DNA,
longer for the analysis of genomic DNA). Additionally, the
amplicons should be less than 500 bp.
5. A reliable real-time PCR reaction should complete following
criteria. First, standard DNA concentrations should cover 7-8
orders of magnitude, such as from 10 8 -10 1 copies of Ad DNA/
reaction. The correlation coeffi cient ( R 2 ) value of the standard
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