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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A quick update on the side project since I'm procrastinating writing a short fellowship grant... About a month ago I realized why my sonications were not working. It turns out that when I moved to Florida from my Graduate work at Iowa, I didn't update my ChIP protocol to reflect a reagent change. At Iowa, we had been using 16% paraformaldehyde as our crosslinking agent. Unfortunately, I was throwing 37% formaldehyde in there instead meaning that I was way over crosslinking my samples which explains why I was having such a hard time shearing the DNA. I practically cemented all of the proteins in the cell together. Anyway, I had some more downtime this week waiting for a new batch of cells to grow for a massive timecourse experiment involving 3 timepoints and 10 different immunoprecipitations for cool proteins. I used this waiting period to optimize sonication conditions with the fancy programmable sonicator.
As you can see, I'm getting the accumulation of a nice smear at exactly the right fragment size, 200bp, for a ChIP-Seq experiment. Maybe in a future post I'll do a write up about how to turn these samples into a multiplexed library for sequencing on a GAIIx or Hi-Seq machine. I'm really not looking forward to sonicating 30 samples and prepping those libraries...
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Glad you got it worked out! Sometimes it comes down to the most (or not so) minute details.
Exactly, I just wish I would have figured this out earlier. It'll be awesome to get this data out of the door.
thanks for sharing the discovery! as obvious as the solutions may seem to us after figuring them out, these trouble-shooting triumphs are so under-shared and always end up being valuable to others!
Raga, I kind of wanted to do this project as an open notebook, but I realized that would take way more effort than it's worth. I'll definitely post my slip ups and mistakes because like you I think there's a lot of value in posting about crap that goes wrong to help others who might be struggling with a similar problem.
I would like to ask the amount of sonicated DNA (in mg or ng) that you loaded in this gel. Thanks.
It's ~1ug total DNA per lane