Sv: Sonication of DNA for cloning

salanti ali at image.dk
Tue Feb 27 05:35:29 EST 2001


Is T4 polymerase really necessary as Klenow also has 3-5
exonucleaseactivity??

Susanne Rohrer <srohrer.nospam at immv.unizh.ch> skrev i en
nyhedsmeddelelse:3A9B7B42.31B32145 at immv.unizh.ch...
>
>
> salanti wrote:
>
> > I am going to make a small library from a 7kb PCR fragment. I plan to
> > fragment the big PCR product by sonication, then treat it with Klenow.
>
> Perhaps it would be a good idea to clone the 7kb Fragment into a vector to
> be able te get enough DNA out of a maxiprep
>
> > But what happens to the "nicks" in the DNA that will occur from the
> > sonication?
>
> they would be "fixed" by Klenow (nick translation). But I don't think
> nicking is a big problem with sonication. (other opinions welcome)
>
> > : Is it OK to sonicate DNA for cloning purposes
>
> yes, it's fine.  you need to add Polynucleotide Kinase and T4 Polymerase
to
> the repair (polishing) reaction as well, eg:
>
> 80ug DNA
> 100uM dNTP
> 1mM ATP
> 10ug/ml BSA
> 3U T4-Polymerase
> 5U Klenow
> 10U PNK
> Total volume of 300ul
>
> 2h at RT
>
> extract with Phenol/Chloroform etc.
>
>
> As I said, other suggestions are welcome
>
> Susanne
>
>
>
>







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