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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Chromatin Immunoprecipitation contamination
Posted by: chavling (IP Hidden, New member, 1)
Date: March 14, 2005 11:16AM
I am getting small amounts of PCR-amplified DNA from my control (pre-immune) reaction when doing chromatin immunoprecipitation/PCR. The positive control (input) and experimental sample (immune serum) are both positive (immune has less DNA amplified), and the negative control for the PCR reaction is flat negative.
I preclear the chromatin samples for 2 hours with pre-blocked Pansorbin cells and then use 1 ul of serum/ ~5x10(7) cells in 0.5 ml volume. I first wash the Pansorbin and then pre-block with 1 mg/ml BSA and 1mg/ml Herring Sperm DNA. Then I wash the pre-blocked Pansorbin cells twice before using for the preclearing step. Anybody have ideas where the contamination is coming from?
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