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Brian Krueger, PhD
Columbia University Medical Center
New York NY USA

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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Thursday, December 30, 2010

I'm beginning a new project in lab.  It's a series of ChIP-seq experiments and the first step to doing ChIP-seq properly is optimizing sonication conditions.  Here's a trial run with the sonicator I plan on using.  The DNA shown in the gel is from cells containing latent herpes virus.  We're looking to shear the DNA so that the bulk of it is between 100 and 600bp.  For ChIP-seq we extract and purify the DNA in the 100-300 range in the gel.  Looks like about 13 cycles of sonication should do (sonication past this point doesn't result in smaller fragments, don't want to risk over sonicating)! Actually, I think I'm going to do this again on Monday.  I looked back at some old data and I should be able to get the fragment sizes a bit smaller by increasing the pulse time.  I'll post an update soon!

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Suzy
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I agree. I think you can get it broken further. Have you ever tried bead beating?


Brian Krueger, PhD
Columbia University Medical Center
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No, never heard of it!  Fancy places have those new Covaris automatic sonicators, but I don't have access to one :(


Suzy
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I am thinking just using 0.1mm glass beads and a vortex to shear it. Or if you have a high powered bead beater like a FastPrep, you could shear the DNA down pretty quickly.

What about using 4 base cutting restriction enzymes?

The sonicator is probably the lease expensive way but the bead beating is easier for multiple samples.


Genomic Repairman
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Jade is right bead beating is the bomb, your samples shall obey.


Brian Krueger, PhD
Columbia University Medical Center
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My cursory google search for ChIP and bead beating turns up a couple of protocols that use it for ChIP in organisms with cell walls.  It's used as a mechanism to lyse the cells, not to shear the DNA.  All of the protocols I found have a sonication or restriction digest step for DNA shearing.


Genomic Repairman
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Here are some good techniques to shear DNA of which I believe bead beating is a part of.

http://linkinghub.elsevier.com/retrieve/pii/S0968000497010803


Dr. Girlfriend
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 Good luck with your project Brian. ChIP was the bane of my lab life! It worked great for one transcription, but not at all for the other two. However, all my problems were related to protein stability, so as long as you can do regular ChIP ok ChIP-seq should work for you. Regards the size, I think you just have to play with your protocol until you get it right (just like you are doing).


Brian Krueger, PhD
Columbia University Medical Center
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Thanks DG! I did a lot of this for my PhD research. I think it'll be fun to do an open notebookish style series on how to do ChIP-seq from start to finish.

Thanks for the link GR. I'll take a look once I can acres the paper. It's behind a paywall :(

Thomas Joseph
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To shear our DNA we used a nebulization method. Worked like a charm if you can get your hands on a nebulization cup and a tank of nitrogen. The protocol I link to says 700-1300bp in size, but you can get it lower by altering viscosity and neb times.

 

http://cshprotocols.cshlp.org/cgi/content/abstract/2006/4/pdb.prot4539


Brian Krueger, PhD
Columbia University Medical Center
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Thanks, TJ. I've heard of nebulization before, but I don't think we have one around.

I re-did the sonication today.  I'm reversing the crosslinks tonight and I should have another cool looking gel tomorrow afternoon.  I really wish I had a 15cm PAGE apparatus in this lab.  The gels look much tighter.


Thomas Joseph
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The "device" itself is very simple. A nitrogen tank with tubing and a nebulization cup. The cups can usually be purchased from any medical store supplier. The nebulizer cups we used to use were from IPI Medical Products, based out of Chicago (part #4101). It needs to be modified slightly, but the protocol can be found here (http://www.genome.ou.edu/protocol_book/protocol_partII.html). I used one cup for my entire grad school career ... cost me around $7 (sans nitrogen tank and nalgene tubing).

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