I have been trying to standardize sonication protocol for HCHO-crosslinked CNS and discs collected from third instar larvae of Drosophila using Bioruptor. I have used discs and CNS from 10 larvae, I do find variations in fragments sizes (either 200-600 bp or 500-1500 bp) using same setting and same reagents. I tried changing several factors e.g. sonication tube, buffer, number of cells, even used different bioruptor. Surprisingly all the samples of each sonication batch produces similar sizes of DNA fragments which cancels the possibility of improper lysis of discs and CNS (I do crush the crosslinked discs and CNS using plastic pestle for eppendorf tubes). Please tell me if you have any idea of whats going on, I have been trying to standardize this for last two months. Thanks...
Hi Sandip,
The position (at the side or middle etc of the unit) of tubes in the sonicator (Bioruptor) also affects outcome. I would use a flat based tube instead of eppendorf. Also, use the same volumes in all tubes and degas the liquid. Sonicate one type of sample at any one time. If your sample is formaldehyde fixed then perhaps check whether the length of time the sample is in fomaldehyde impacts on your fragment size.
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