Brian Krueger is the owner, creator and coder of LabSpaces by night and Next Generation Sequencer by day. He is currently the Director of Genomic Analysis and Technical Operations for the Institute for Genomic Medicine at Columbia University Medical Center. In his blog you will find articles about technology, molecular biology, and editorial comments on the current state of science on the internet.
My posts are presented as opinion and commentary and do not represent the views of LabSpaces Productions, LLC, my employer, or my educational institution.
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I've been working on this project on and off for a few months now. You may remember my previous post about attempting sonication trials. Unfortunately, things in lab have been busy trying to get a new graduate student started and troubleshooting problems with other experiments so I haven't had as much time to devote to this project as I would like. I've spent the past month or so trying to optimize my sonication conditions on a sonicator that's in my building, and I've gotten less than spectacular results (I've tested at least 10 different conditions on this machine ex: buffers, cell densities, sonication intensity, sonication duration). I'm looking to break my DNA up into short 100-300 base pair fragments and previously I was only able to get them down to about 600. I decided it was time to test other sonicators and here are the results.
Fisher 100 sonic dismembrator vs Misonix 4000 - 1/8" tapered tip.1 mL of RIPA buffer containing 1x107 cells. Numbers above the lanes indicate the number of cycles that were done (30 seconds on 1.5 minutes of rest between cycles)
It looks like the Fisher 100 is the way to go. Although it's a completely manual system. I think I could probably get the Misonix to the same level of sonication and the nice thing about that machine is that it's programmable, there aren't any knobs to turn and I don't have to sit there for an hour manually turning the machine on and off. I have enough cells left to do one sequencing run (for my 6 proteins), so I think I'm going to do an experiment with the Fisher machine and then on my next set try to get the Misonix working better so I have that extra added level of experimental consistency. I contacted an old collaborator of mine who used the same Misonix system for his experiments and he has given me his conditions (He used a power level of 7, which could make a huge difference). I will say that I'm not all that happy with the amount of shearing. When I did similar trials in graduate school I was able to completely get rid of the high molecular weight DNA and get a nice smear in the 100-500 range. However, after looking through many sonication protocols, these results are not all that different from what other labs are using as input for their ChIP-seq experiments. I just wish we had a Covaris available here :(
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Just a follow up, but I was perusing my protocol today and noticed that in my old lab we started using 16% paraformaldehyde (some think it's a better fixing agent) to make the 11% fixing solution. In these sonication trials I had been using 37% formaldehyde, but my recipe was created calculating this as 16%, so instead of using 11% fixing solutions it was more like 25%... This makes cells super hard to bust open. So, here's the lesson, always double check your protocols and recipes after switching labs.