Adding a polyA tail to a vector?????

John Watson watson_j at bms.com
Tue Jul 8 19:37:28 EST 1997


> 
> Greg Nattrass writes:
> 
> > I need to generate a polyA tail of approx 70 adenosines for a Xenopus
> > expression vector. Would Terminal deoxynucleotidyl Transferase (TdT) be
> > the enzyme to use for this application. I have used it to tail cDNA but
> > am unsure as to how I should use it on plasmid DNA.

_snip_
> 
To which Dom Spinella replied:
> 
> Well you could use terminal transferase, but my impression is that
> homopolymer purine tails spontaneously terminate at about 20 residues
> (this is certainly true for polyG tails, and I believe true of polyA
> tails as well -- regardless of the dNTP or TdT concentration).  However,
> it is not clear to me why you would want to do this for your
> application.  Why not just use an expression vector with a
> polyadenylation signal, and let the transfected cells do the work for
> you?

Unfortunately, you don't transfect Xenopus oocytes; you make cRNA and
microinject it. That's why he wants the poly A tract. I will ask my
colleague, who often makes cRNA for Xenopus expression, to see what they
have done.

AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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