EpiTect ChIP qPCR Assays

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Warm IP Lysis Buffer to room temperature to prevent precipitation. -Prepare one extra plate of cells to estimate cell number. -Thaw Protease Inhibitor Cocktail (PIC) at room temperature. -It is recommended to take some trial to optimize sonication condition before beginning with sonication.	-In general it is recommended that one million mammalian cells are required for each IP fraction. -Centrifuge the sample at 6000 × g for 5 min at 4 °C to eliminate bubbles. -To reduce background, remove as much supernatant as possible in the last wash. Continue with next step OR store at 4°C overnight.

Upstream tips
-Warm IP Lysis Buffer to room temperature to prevent precipitation. -Prepare one extra plate of cells to estimate cell number. -Thaw Protease Inhibitor Cocktail (PIC) at room temperature. -It is recommended to take some trial to optimize sonication condition before beginning with sonication.
Protocol tips
-In general it is recommended that one million mammalian cells are required for each IP fraction. -Centrifuge the sample at 6000 × g for 5 min at 4 °C to eliminate bubbles. -To reduce background, remove as much supernatant as possible in the last wash. Continue with next step OR store at 4°C overnight.

Publication protocol

Fixing and preparation for immunostaining: NIH3T3 cells were grown on rectangular patterns for 3 hr, 3 days, 6 days, and 10 days. The cells at the four-stated
time-points were fixed with 2% formaldehyde for 5 min at room temperature (RT). Following fixation, cells were quenched with 127 mM glycine for 10 min at RT. Cells were then washed with Phosphate-buffered Saline (PBS). The cells were lysed and nuclei were prepared in lysis buffer (10 mM Tris-HCl (pH 8), 10 mM NaCl, 0.2% IGEPAL CA-630 (Sigma)) with protease inhibitor cocktail (Roche) for 30 min on ice with intermittent agitation. Nuclei were washed with 1x Fast Digest (FD) buffer (Thermo Fisher Scientific). 400 μl of 1 x FDbuffer and 6 μl
of 20% SDS was added to the nuclei and incubated at 37˚C for 60 min with constant agitation.

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Manufacturer protocol

Download the product protocol from Qiagen for EpiTect ChIP qPCR Assays below.

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