[Zbrafish] Large bands in in-situ probes

Burdine, Rebecca D rburdine at Princeton.EDU
Tue Aug 29 09:19:59 EST 2006


Hi Catherine,

Are you treating your reaction with DNAse after the RNA transcription?
If not you will see a faint band; probably around 3kb if you are using
bluescript plus a 450bp insert.

Some people do the DNAse step and others do not so it may not be in your
protocol.

Becky

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Rebecca D. Burdine, Ph.D.
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-----Original Message-----
From: zbrafish-bounces at oat.bio.indiana.edu
[mailto:zbrafish-bounces at oat.bio.indiana.edu] On Behalf Of catsglynn
Sent: Tuesday, August 29, 2006 9:10 AM
To: bionet-organisms-zebrafish at moderators.isc.org
Subject: [Zbrafish] Large bands in in-situ probes

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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