long polyA after using polyT17 primer - why???

Domo via methods%40net.bio.net (by rimask from yahoo.com)
Sat Oct 28 03:16:38 EST 2006


Hello,

I have some mistery in experiments. Maybe someone has clue or experience =
about that.

I am doing cDNA library construction.

1. RNA is treated by DNaseI (to remove DNA), then mRNA is selected =
(Qiagen kit).
2. After dephosphorylation and decapping, I ligate RNA oligo.
3. Then first strand cDNA synthesis is performed by polyT17 primer.

AACGTGGCTCGAGTGGCTGAACGCTTTTTTTTTTTTTTTTTVN

or=20

AACGTGGCTCGAGTGGCTGAACGCTTTTTTTTTTTTTTTTTV

After PCR (=3D second strand synthesis) and restriction digestion of =
ends, the product is purified by Amersham SB-400 column (or gel) and =
cloned to vector.

To my surprise, after sequencing, I get a little of insert (8-80 bp) and =
long TTTTT afterwards (50 - 250 bp).

As I assume, the polyT primer should bind to very beginning of mRNA =
polyA tail and would expect only 17nt of polyT, but not hunderts.

Where does come these long polyT from? Any suggestions?

Thank you!



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