In vitro transcription of a large mRNA

Hinayana Bawagan bawagan at umdnj.edu
Sat May 7 00:31:03 EST 1994


dm690785 at mbcr.bcm.tmc.edu (Derek Milner) writes:

>I am about to embark upon a project designed to determine if a neuronal mRNA
>contains a "zipcode" sequence which specifies its subcellular localization.
One
>of the experiments I plan to do is to synthesize the mRNA in vitro, label
by
>fluorophore conjugation, microinject into cultured neurons and observe
transport
>of the mRNA by laser scanning confocal microscopy. My question is this: The
>transcript is rather large ( approx 9kbp). Can a transcript of this size be
>synthesized by in vitro transcription? I have seen a reference in which a 
>viral RNA of slightly larger than 7 kb has been synthesized by in vitro
>transcription. Does anyone know if there is a limit to the size of an RNA
that
>can be synthesized by in vitro transcription, and does anyone have any hints
or
>advice to give concerning in vitro transcription of large RNAs?

>Thanks,

>-Derek
    
    The following may be useful to you:

1. Krieg, Paul A. "Improved synthesis of full-length RNA probe at reduced
incubation temperatures." Nucleic Acids Research, Vol 18, No 21 p. 6463.

2. If you are using SP6 pol:
   Krieg, P.A. and D. A. Melton. "In vitro RNA Synthesis with SP6
Polymerase." Methods in Enzymology Vol 155, p. 397-415.

3. The Promega book on protocols. Also the Promega technical manual on
Transcription in vitro systems.

Good luck!



More information about the Methods mailing list