banner
You are not using a standards compliant browser. Because of this you may notice minor glitches in the rendering of this page. Please upgrade to a compliant browser for optimal viewing:
Firefox
Internet Explorer 7
Safari (Mac and PC)
Post Archive
2024 (0)
2015 (3)2014 (1)2013 (4)2012 (5)2011 (21)
October (8)August (3)July (1)

More Troubleshooting
Wednesday, July 13, 2011
June (1)

End to the sonication saga
Tuesday, June 7, 2011
May (1)April (2)March (3)

Circle of life
Thursday, March 17, 2011

Curing a plague: Cryptocaryon irritans
Wednesday, March 9, 2011

Video: First new fish in 6 months!!
Wednesday, March 2, 2011
February (1)January (1)
2010 (13)
December (3)

The first step is the most important
Thursday, December 30, 2010

Have we really found a stem cell cure for HIV?
Wednesday, December 15, 2010

This paper saved my graduate career
Tuesday, December 14, 2010
November (3)

Valium or Sex: How do you like your science promotion
Tuesday, November 23, 2010

A wedding pic.
Tuesday, November 16, 2010

To rule by terror
Tuesday, November 9, 2010
October (2)September (5)

Hiccupping Hubris
Wednesday, September 22, 2010

A death in the family :(
Monday, September 20, 2010

The new lab fish!
Friday, September 10, 2010

What I wish I knew...Before applying to graduate school
Tuesday, September 7, 2010

Stopping viruses by targeting human proteins
Tuesday, September 7, 2010
Rate This Post
Total votes: 2
Blogger Profile

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.

Blog RSS Feed
RSS Add to My Yahoo Add to Google
Recent Comments

Jaeson, that's not true at most places.  Top tier, sure, but 1100+ should get you past the first filter of most PhD programs in the sciences. . . .Read More
Jun 24, 2013, 8:39am

All I can say is that GRE's really do matter at the University of California....I had amazing grades, as well as a Master's degree with stellar grades, government scholarships, publication, confere. . .Read More
Jun 19, 2013, 11:00pm

Hi Brian, I am certainly interested in both continuity and accuracy of PacBio sequencing. However, I no longer fear the 15% error rate like I first did, because we have more-or-less worked . . .Read More
Feb 26, 2013, 12:13am

Great stuff Jeremy!  You bring up good points about gaps and bioinformatics.  Despite the advances in technology, there is a lot of extra work that goes into assembling a de novo genome on the ba. . .Read More
Feb 25, 2013, 10:20am

Brian,I don't know why shatz doesn't appear to be concerned about the accuracy of Pacbio for plant applications. You would have to ask him. We operate in different spaces- shatz is concerned a. . .Read More
Feb 25, 2013, 8:01am
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!

This post has been viewed: 2747 time(s)

Blog Comments

Suzy
Rate Post:

Like 0 Dislike

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


Brian Krueger, PhD
Columbia University Medical Center
Rate Post:

Like 0 Dislike

No, never heard of it!  Fancy places have those new Covaris automatic sonicators, but I don't have access to one :(


Suzy
Rate Post:

Like 0 Dislike

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
Rate Post:

Like 0 Dislike

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


Brian Krueger, PhD
Columbia University Medical Center
Rate Post:

Like 0 Dislike

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
Rate Post:

Like 0 Dislike

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
Rate Post:

Like 0 Dislike

 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
Rate Post:

Like 0 Dislike
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
Rate Post:

Like 0 Dislike

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
Rate Post:

Like 0 Dislike

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
Rate Post:

Like 0 Dislike

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).

Add Comment?
Comments are closed 2 weeks after initial post.
Friends