[Zbrafish] Re: making long mRNA transcripts

pavlina.haramis from gmail.com via zbrafish%40net.bio.net (by pavlina.haramis from gmail.com)
Tue Jun 10 02:48:05 EST 2008


On Jun 9, 6:21 pm, michelle.em... from gmail.com wrote:
> Hello,  I have been recently trying to transcribe GFP- or myc-tagged
> mRNA to attempt rescue experiments using long templates (3-4 kb).  I
> believe that my transcription reactions are working, but that the
> transcripts are ending prematurely (my tags are on the 3' end, and I
> am not seeing expression).  I do see a nice, tight band on a gel at
> about half the length of the template, and a very faint band at the
> correct size.  I am using pCS2+, SP6 enzyme, and mMessage mMachine,
> and running the reaction for 2-2.5 hr.   Ambion says that these are
> short enough transcripts to work with the kit, and their handbook says
> it should be good for up to 5 kb.  However, something isn't working
> for me, and I've been trying multiple templates.  Has anyone had
> success using this kit for making mRNA in this size range?  Thanks
> very much for your suggestions!  --Michelle

Hi Michelle,
We have a similar problem and I can not really be of much help only to
let you know that what helped in one of our cases was that we should
absolutely include the SV40 polyA tail in the transcript. In one case
that made all the difference between no expression ( of the 3' tag) to
strong expression visible at 24hpf. Unfortunately though even with the
SV4o tail we still do not get expression of some other long
transcripts so I am also keen to hear suggestions!
Cheers
pavlina



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