ChIP DNA sonication conditions - (Oct/18/2005 )
I need to sonicate a DNA sample from a chromatin immunoprecipitation into 200 bp fragments, but I do not really know which must be the sonication conditions.
I do not get fragments less than 2000 bp. I am using a Vibra Cell from SONICS. I would need the output watts, the amplitude and number and timing of cycles to sonicate the ADN in this model.
Thank you! :-)
DNA sonication conditions have been frequently discussed here. Do a search and you will find out.
Many variables affect sonication such as the type of machine and its settings, volume, type of cells, type of tubes, etc. You have to test different conditions to see which give you the best results before you rush to real ChIP assays.
Agreed. This is the most important step. Try different settings because it will vary from machine to machine. You could try contacting the machines company and ask them which settings they suggest.
I think the best way to find out is to do titration of sonication pulses (one variable at the time, like first amplitude, then percent duty, and time; assumed that the vol is consistent--for me around 2.5mL). You need to collect a small amount at each pulse to decross-link to check the fragment size. You may try to add MNase enzyme, which digest between nucleosome.