RNA polyA tail

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Fri Jun 15 10:03:45 EST 2001


In <001a01c0f4d9$fd24c5a0$7566c641 at brnijhu.org>, dottor wrote:
>

>Dear all,
>
>i would like to know if anybody knows how to introduce in a DNA 
template =
>within a vector  ( and which should be the specific sequence) a DNA =
>sequence that allows to have in the corrresponding RNA transcript a 3' =
>polyA tail of about 60 bases.

I did it a long time ago once, and it was pain. Here is the outline: say you 
want to add poly(a) to site "A." Cut the vector with "A" and divide into two 
batches. Use terminal transferase to add A's on one batch and T's on 
another batch. Then cut each batch at a second site "B." Gel purify on 
fragment that is A tailed from one batch and the other fragment that is T 
tailed from the other batch and ligate.

I did an initial screen by RE sites flanking the polyA and running the digest 
on an acrylamide gel. Definitely sequence the ones you think are good, 
because I got a lot of "surprises." You may not be able  to tell exactly how 
long the A-run is, because the sequencing reaction poops out, but you 
can get an estimate.

If you are trying to make poly(A) RNA in vitro, there is also a way you can 
add poly(A) after transcription as well.

Nick

-- 
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu

 




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