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
2017 (0)2015 (3)2014 (2)
August (1)

TwitterOauth API delete tweets
Wednesday, August 27, 2014
January (1)

Illumina's $1000 Genome*
Wednesday, January 15, 2014
2013 (4)2012 (6)2011 (21)
October (7)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 (4)

Thwart the NYtimes paywall
Thursday, March 17, 2011

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: 0
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

The Bowflex Extreme SE is a great piece of home gym equipment. It offers a huge range of exercises that hit every single muscle group of the body, get Read More
Jun 28, 2017, 2:21am

Save your money, TreadClimber and Max Trainer cardio machines, Bowflex home gyms, Bowflex SelectTech dumbbells and more. Bowflex is your solution, get Read More
Jun 28, 2017, 12:48am
Comment by jasa pembuatan website in Antineoplastons? You gotta be kidding me!

Anda yang memiliki toko online sebaiknya mulai memikirkan keuntungan menggunakan jasa pembuatan website toko online.Meskipun perlu mengeluarka dana untuk . . .Read More
Jun 28, 2017, 12:41am

I must appreciate the way you have expressed your feelingsthrough your blog!.. run 3 . . .Read More
Jun 26, 2017, 12:50pm

Interesting article! Thank you for sharing them! I hope you will continue to have similar posts to share with everyone! fb login . . .Read More
Jun 26, 2017, 3:27am
Tuesday, June 7, 2011

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.


Misonix 4000, power 60, 30s on 1 min off. Numbers above lanes equals the number of cycles

 

 

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...

This post has been viewed: 1500 time(s)

Blog Comments

Genomic Repairman
Rate Post:

Like 0 Dislike

Glad you got it worked out!  Sometimes it comes down to the most (or not so) minute details.


Brian Krueger, PhD
Columbia University Medical Center
Rate Post:

Like 0 Dislike

Exactly, I just wish I would have figured this out earlier.  It'll be awesome to get this data out of the door.


Ragamuffin
Rate Post:

Like 0 Dislike

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!


Brian Krueger, PhD
Columbia University Medical Center
Rate Post:

Like 0 Dislike

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.

kinder_ovni

Guest Comment

I would like to ask the amount of sonicated DNA (in mg or ng) that you loaded in this gel. Thanks.


Brian Krueger, PhD
Columbia University Medical Center
Rate Post:

Like 0 Dislike

It's ~1ug total DNA per lane

 

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