Nebulization

[snorider]

I cannot get dna to fragment to 200bp. I am using nebulization
protocol from Illumina.
I have tried 35psi 6 minutes, 35psi 8 min, 35 psi 10 min, 37psi 6 min,
& 40psi 6 min. I am trying this on dna fragments ranging from 10 kb to
0.5 kb. I then run them on a 2% agarose gel and I get a smear between
300 & 500 bp. Any suggestions?
Thanks

[kwalton]

Hi!
I was in the same situation. I never did get nebulization to work the
way Illumina says it will. After 2 months of troubleshooting their
protocol, doing many of the same things you've done as well as some
other things, it never worked. I submitted to Sonication. I used both
the Sonicator 300 and the Bioruptor. I found that with the Bioruptor
you can do many 1.5mL tubes at one time without contamination being an
issue. (consistancy is also better) The Sonicator 300 was a single
probe that could potentially cause contamination. If you would like
information on the Bioruptor I can get it for you. I'm sure other
sonicators would work just as well just be careful of single probe
sonicators unless you have a fool proof cleaning method. I brought up
5ug of DNA in 50ul of TE then added 150 ul of Illumina's Nebulization
buffer to the tube. I sonicated it on High for 30 sec ON and 30 sec
OFF for a total of 30 min and then cleaned it with a Qiaquick protocol
that follows nebulization in the Genomic Prep protocol from Illumina.
You should find that sonication will give the correct sizes. If you
would like more info feel free to email me. Hope this helps!

[Kittler, Ellen Ph. D.]

I would love some info about the sonicator you chose!
The BioRuptor ??

Ellie

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Ellen LW Kittler, PhD
CFAR Core Services Coordinator
UMass Center for AIDS Research, and
Program in Molecular Medicine
UMass Medical School
373 Plantation Street, Room 207
Worcester MA 01605
(508) 856 - 6137 phone
(508) 856 - 6187 fax

[kwalton]

It is the Bioruptor from Diagenode out of Belgium.
www.diagenode.com

From the .pdf

The Bioruptor System is based on a water bath with high power
ultrasound generating elements located below the tank.
With the Bioruptor, 6 to 12 closed tubes can be sonicated together and
the continuous rotation of tubes allows even distribution of the
energy.
With a better control on the parameters, the Bioruptor enables the
automation of the sonication step which guaranties higher
reproducibility and constant results.
The frequency of the ultrasound energy produced by the Bioruptor and a
probe sonicator is equivalent (20 kHz).

What is the effect of ultrasound on biological samples?
A generally accepted view is that ultrasound produces a gaseous
cavitation in the liquid. This term describes the formation of small
bubbles from dissolved gases or vapors due to alteration of pressure.
These bubbles are capable of resonance vibration and produce vigorous
eddying or microstreaming, which is sufficient to break cells. Also,
the fragmentation of DNA takes place as a consequence of mechanical
stress or shear from the bubbles.
With a probe sonicator, the microstreaming phenomenon is limited to
the vicinity of the probe, whereas for the Bioruptor, the whole volume
of water present in the tank is exposed to ultrasound energy.

I can send the .pdf to individual accounts if anyone would like to
look it over.