DNA shearing enzyme

[Gorbenzer]

Hi everyone,
anyone know what's the enzyme used for the DNA share step of library preparation?

We have problems finding the correct exposition time for our bacteria. The 15 min suggested cuts too much and it cuts too much.

[IonTorrent]

Hi everyone,
anyone know what's the enzyme used for the DNA share step of library preparation?

We have problems finding the correct exposition time for our bacteria. The 15 min suggested cuts too much and it cuts too much.

Hi Gorbenzer,

While the recommended shearing time for each library insert size is a great starting point, there are cases where sample integrity or other attributes necessitates optimization of this parameter. High protein carryover is one factor that can lead to overshearing, and EDTA > 0.1mM, undershearing.

Assuming you are using the current Ion Xpress™ Plus gDNA Fragment Library Preparation Kit, there is more information available on pp. 15-18 of the User Guide:

http://lifetech-it.hosted.jivesoftware.com/docs/DOC-1997

For more guidance I would also recommend contacting your local Field Applications Scientist.

[ozzy]

using an enyzme other than kits shearing enzyme

Hi everyone,
We sequence pcr amplicon, so we know the amplicon size and sequence. We have problems with shearing and i think that we can use an endonucleases instead of kit’s shearing enzyme.
Do you have such an experience or an idea about DNA sheaaring step other than currently used?
Thanks

Özlem

[ozzy]

Hi Genseq,
I want to make sure the size of DNA fragments by using endonucleases. I’m interested in 200-250bp fragments. But these enzymes are randomly cutting, i need to do optimization with these. But i think if i use restriction endonucleases (for example EcoRI) i know exactly what the size range of fragments. Isn't it?
Thanks

[ozzy]

your answers really help me
thanks a lot

[jimmyinfrance]

Genseq, which one would you recommend/had experience with?

http://www.epibio.com/item.asp?ID=325

http://www.ribosolutionsinc.com/cyanase1.html

http://www.sigmaaldrich.com/catalog/...g=en&region=RU