long polyA after using polyT17 primer - why???
(by rimask from yahoo.com)
Sat Oct 28 03:16:38 EST 2006
I have some mistery in experiments. Maybe someone has clue or experience =
I am doing cDNA library construction.
1. RNA is treated by DNaseI (to remove DNA), then mRNA is selected =
2. After dephosphorylation and decapping, I ligate RNA oligo.
3. Then first strand cDNA synthesis is performed by polyT17 primer.
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?
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