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  • Odd fragmentation patterns using a Bioruptor for sonication

    I am trying to create a genomic library for transposon integration profiling in yeast.
    Recently I have been getting a very strange fragmentation pattern. It seems that my fragmentation is at the same time too high and too low.
    I am currently shearing for 13 cycles, 3 sets of 3, and one set of 4 for 30 seconds on and 30 seconds off. Others using this protocol for different organisms have gotten great fragmentation with a peak at ~350bp, which is what I am aiming for. I have seen this in the last few samples Ive been using to optimize my procedure. At first I thought that maybe consecutive freeze thaws or DNase contamination may be the problem, but even using samples isolated from the night before and immediately put into the -20 showed this fragmentation, despite two attempts.

    A quick rundown of my protocol is using zymolase then the epicenter kit to break open the cells and precipitate most of the protein. From there I treat with RNase A for 1 hr, proteinase k for 2 hours, a phenol chlor extraction, chlor back extraction, and finally an ethanol precipitation. The sample on this BioA had 6 ethanol washes in the ethanol precipitation.

    Attached is a BioA of my most recent attempt. the first 8 samples are mine the others belong to someone who was sharing the run with me.
    The key is the first 4 samples are from one isolation at 300-600 ng of total DNA increasing by 100 ng per sample. The second 4 samples are the same setup just from a different isolation.

    If anyone has any advice on how to improve my fragmentation or an explanation of what could be causing the strange fragmentation pattern I would be very appreciative.
    Andrew
    Attached Files

  • #2
    I have just started doing RNAseq and had to use Bioruptor Pico for fragmentation; I only had 4 samples to test the protocol prior to the real deal (I aimed at 300bp fragments). In the protocol for Bioruptor they point out that you should spin briefly halfway through the sonication if you are aiming at larger fragments (>300bp). I split my 4 samples into two groups so I sonicated one group without spinning halfway through and the other group with the short spin. I saw something quite similar to your Bioanalyser High Sensitivity DNA traces in the group that wasn't spun (don't have the pdf on me atm). The group with short spin looked perfect in all runs.

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