long polyA after using polyT17 primer - why???

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

AACGTGGCTCGAGTGGCTGAACGCTTTTTTTTTTTTTTTTTV

After PCR (= 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!

degraded primers / exonuclease contamination?

Wo


Domo wrote: