What's in Megascript buffer ?

Patric Lundberg psl at ahabs.wisc.edu
Wed Oct 12 23:59:46 EST 1994


In article <941013.094559.PDT.MUNGO at UVVM.UVic.CA>, MUNGO at UVVM.UVIC.CA
wrote:

> Does anyone know what the secret behind Ambion's megascript buffer is ?
> I gather these buffers work extremely well, and the "magic" ingredient
> isn't polymerase or nucleotides, so what is it ?  Along the same lines
> why is it so expensive (other than it works) ?
> Mungo

I suspect there may be several alterations to their protocol.  One thing in
particular that they probably have changed is the _type_ of buffering agent
they use.  Most in vitro transcription protocols call for using Tris to
buffer the reaction - remember that a proton is released every time a
nucleotide is added to your RNA.  Consequently, a really high capacity
buffer that can maintain the original pH would be desired.  Tris is not a
very potent buffering agent.  I routinely use HEPES instead of Tris, and
get 5-10 times more RNA/transcription.  The fact that HEPES works better
than Tris is no secret; Novagen uses HEPES in some of their
transcription/translation kits, and a group of Russian researchers (can't
locate my ref right now) did a comprehensive study on the effect of various
salts, buffer conditions and enzyme concentrations of the efficiency of T7
and SP6 transcription.

My suggestion would be to make your own 10X buffer w/HEPES, and to play
around with all salt concentrations, incubation temps (35-42'C), and
absolutely the amount of template DNA.
Also, any residual amount of solvent from the DNA purification step such
EtOH and phenol can virtually eliminate transcription - I always spin my
linearized DNA template over Microcon-100 membranes before use.


I found all of the above to drastically affect my transcrition
efficiencies.

Sincerely, Patric (and all those disclaimers).

< -------------------------------------------------- >
  psl at ahabs.wisc.edu
  UW-Madison, Mad-town USA

  Ma'let a"r ingenting, va"gen a"r allt - R. Broberg
< -------------------------------------------------- >



More information about the Methods mailing list