dG tailing of mRNA

Wendy-Anne Smith wendy at ichr.uwa.edu.au
Tue Apr 22 01:37:23 EST 1997


I am trying to add a string of G's to the 3'end of my DNA after 1st strand
synthesis.  I am using total RNA not poly mRNA primed with oligo dTfor
forst strand synthesis and that is working fine.  Then i take an aliquot
(1ug) of the original total RNA and use that directly in a tailing
reaction.  I have been setting up a 25ul reaction with a final [] of 0.4mM
dGTP and 25U of TdT.  I then incubate the reaction at 37C for 30-45 min,
and ppt the reaction with isopropanol.  The sample is then resuspended and
2 pcr's performed one with internal primers and one with the primer
corresponding to the dG tail.  The internal primer reaction works well and
the other doesn't work.  So i don't know whether my tailing reaction isn't
working or my PCR.  For the PCR i am following from a paper but
unfortunately the paper is not very clear.

I was wondering whether anyone had tried a similar expt and could provide
any advice.


Wendy-Anne Smith

TVW Telethon Institute for Child Health Research
(Company Limited by Guarantee)
Western Australia

Fax 61 9 388 3414

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