[Zbrafish] making long mRNA transcripts

michelle.emond from gmail.com via zbrafish%40net.bio.net (by michelle.emond from gmail.com)
Mon Jun 9 11:21:26 EST 2008


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



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