Hydroshear DNA

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Tue May 27 19:19:37 EST 2008


On May 27, 11:50 am, d... from no.email.thankstospam.net (DK) wrote:
> In article <53307375-5f42-439d-8128-9cbf5f01a... from m45g2000hsb.googlegroups.com>, peter <peter.ianak... from gmail.com> wrote:
>
>
>
> >On May 26, 2:44 pm, "Jose de las Heras" <jose... from tiscali.co.uk> wrote:
> >> "peter" <peter.ianak... from gmail.com> wrote in message
>
> >>news:5aa1d425-dcae-4ab1-8a6a-4b3d6a0a7e7b from k13g2000hse.googlegroups.com...
>
> >> > Hello guys,
> >> > I need some expertise in random DNA fragmentation. I was thinking of
> >> > hydroshearing DNA by passing trough syringe. Does anyone know the
> >> > diameter of the needle needed to shear the DNA? is there better
> >> > methods? - any experience is greatly appreciated.
> >> > Thanks,
> >> > Peter
>
> >> We used to "autoclave" (actually using a standard kitchen pressure cooker)
> >> wheat genomic DNA to fragment it. This was many years ago in another life
> >> and I have never tried that again. I can't remember the size range we
> >> obtained... but if you don't have a sonicator available (which would be the
> >> best option) it should be a simple thing to try and see if it suits you. I
> >> suspect you may find most of it is above the 0.5-2kb range you wish... but I
> >> just don't remember.
>
> >> Jose
>
> >Boiling (denaturing) is not an option, sorry. I need to clone, make
> >libraries.
>
> Limited DNAse digestion. Fix time, titrate DNAse, quench with EDTA,
> heat inactivate, run gel, excise and purify needed MW range, blunt by
> filling in with Pfu poly, ligate, transform.
>
> DK

DK Interesting thought about the DNAse-Pfu combination, do you know
where are the phosphates after DNase treatment?
Thanks
Peter


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