[Zbrafish] Re: Zbrafish Digest, Vol 25, Issue 8

Wilson Clements via zbrafish%40net.bio.net (by wclements from ucsd.edu)
Fri Jun 29 20:00:24 EST 2007

Dear Sarah,

With respect to your probe synthesis problems, here are some things  
to try:

1)  Make sure that the enzyme you are linearizing the template DNA  
with is an enzyme that leaves a 3' overhang and not a 5' overhang.   
RNA polymerases wrap around the end of the template in the presence  
of a 5' overhang, so you will get multiple longer extension products  
consistent with the higher molecular weight bands you describe.
2)  (Sounds like you already did this, but just in case) To get rid  
of secondary structure, put your gel samples in a formamide-based RNA  
loading buffer (e.g. from Ambion), heat at 65C for 5', at the same  
time, pre-run your gel for 5'.  Put your heated samples on ice and  
load immediately.

Good luck.
Wilson Clements, Ph.D.

wclements from ucsd.edu

Dept. of Biology
Section of Cell and Developmental Biology
University of California at San Diego
9500 Gilman Dr.
Natural Sciences Building 6105
La Jolla, CA 92093-0380

TEL    (858) 534-6955
LAB    (858) 822-4658
FAX    (858) 822-5740

On Jun 29, 2007, at 10:01 AM, zbrafish-request from oat.bio.indiana.edu  

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> Date: Thu, 28 Jun 2007 10:42:37 -0700
> From: sarah at RIC <sspinette from ric.edu>
> Subject: [Zbrafish] probe synthesis problems?
> To: bionet-organisms-zebrafish from moderators.isc.org
> Message-ID: <1183052557.780101.71200 from k79g2000hse.googlegroups.com>
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> We are starting up some in situ expereiments. We are using a Ntl
> plasmid as a control along with our sequence of interest (which is
> about 750nts). We ran some of our antisence and sence probes on a gel.
> The ntl probes both look like beautiful sharp bands of the right size
> with no degredation detected. However, in the lanes in which we ran
> our probes of interest we see a faint band of the right size but we
> also see a bunch of larger bands as well ranging from ~1000nt-2500nt.
> we checked that our plasmids ere fully linearized with the right
> enzyme. We are wondering if it could be secondary structure that is
> resistant to 65 deg incubation or some other problem...please help!
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