EpiTect ChIP OneDay Kit

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

ChIP assays were performed with the EpiTect ChIP Oneday kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. A total of 5 × 106 MIA Paca-2 cells were cross-linked with 1% formaldehyde at room temperature for 10 min. Sonication was performed on ice to get 200 to 1,000 bp DNA fragments. Chromatin was pre-cleared with anti-IgG antibody (Santa Cruz Biotechnology) and immunoprecipitated with an anti-SP1 antibody. After reverse cross-linking and DNA isolation and purification, DNA from input (diluted 1:100) or immunoprecipitated samples were assayed by quantitative PCR (qPCR), performed on a LightCycler®480 Real-Time PCR system (Roche, Diagnostics, Mannheim, Germany) using the SYBT Green I Mastermix (Applied Biosystems). The products were then separated by 2% agarose gel electrophoresis. The following primers were used: P1 (−898 to −778, F: 5′-CAACCCTGACCCCAGCCTCT-3′, and R: 5′-GCAAGGAAAGAGAC ACAGCA-3′), P2 (−878 to −758, F: 5′-GTCCTTTACTCCACAGATAGAT GGGT-3′, and R: 5′-GGGACAACTATTTGGCTGCAG-3′), P3 (−759 to −639, F: 5′-CTGCGCCAAATAGTTGTCCC-3′, and R: 5′-AGCAGGGCTTAGAG AGAAACG-3′), P4 (−568 to 448, F: 5′-GGACATCTGGGGGTCCTCT-3′, and R: 5′-GCGGATTTTGCATTTCACTCCA-3′), P5 (−210 to 90, F: 5′-GTTGC CCAGAGCTCACTG-3′, and R: 5′-GGTTACCCTACTGGCCTGG-3′).

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Manufacturer protocol

Download the product protocol from Qiagen for EpiTect ChIP OneDay Kit below.

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