I am working with liver samples and I am having aggregate formation after the SDS/triton x-100 incubation steps previous to first restriction. Im working with 1x10-7 cells and Im filtering my cells in a 70um cell strainer after dounce homogenizing my samples. My current lysis buffer is prepared in a final volume of 5ml, 10mM Tris HCl pH 8, 10mM NaCl, 0.2 % NP-40 and protease inhibitors and the incubation time is 1 hour in rotation at 4oC.
I use the same buffer for 3C (I am going to do 4C), and I sometimes also have quite big snowflakes-like nuclei aggregates after SDS/triton addition. It shouldn't affect the followingsteps. Try to gently homogeneize the nuclei and to handle them very gently. If still occuring, it could be that you use too many nuclei (see note and trouble shooting sections of these published protocols :
- Article 4C-seq from start to end: a detailed protocol for sample pre...
- Article Multiplexed chromosome conformation capture sequencing for r...
)Hope it will help !
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