[Zbrafish] Re: Large bands in in-situ probes

Dave Parichy dparichy at gmail.com
Wed Aug 30 09:37:59 EST 2006

Sac I and Sac II leave a 3' overhang when they cut, which sometimes can
lead to the polymerase switching strands and producing a larger
transcript (Kpn I and Pst I can have this problem too).  You might try
cutting with a different enzyme that doesn't leave a 3' overhang.

Dave Parichy

catsglynn wrote:
> Hi,
> I'm trying to make anitsense and sense probes (450bp) for in-situ
> hybridizations but I'm frequently getting a larger band of about 3kb in
> size. I have used SacI and SacII to digest the insert from PGEMTeasy,
> and it has digested completely. However, I keep getting a larger band,
> either in the sense or antisense probe. Has anyone encountered this
> before? Can anyone help me please?
> Thank you,
> Catherine.
> Catherine Glynn, M.Sc.,
> Lucy Byrnes' Lab,
> Department of Biochemistry,
> National University of Ireland, Galway,
> Ireland.

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