long polyA after using polyT17 primer - why???

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Sat Oct 28 14:07:30 EST 2006


degraded primers / exonuclease contamination?

Wo


Domo wrote:
> 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!
>
>
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> <DIV><FONT face="Courier New">Hello,</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">I have some mistery in experiments. Maybe someone
> has clue or experience about that.</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">I am doing cDNA library construction.</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">1. RNA is treated by DNaseI (to remove DNA), then
> mRNA is selected (Qiagen kit).</FONT></DIV>
> <DIV><FONT face="Courier New">2. After dephosphorylation and decapping, I ligate
> RNA oligo.</FONT></DIV>
> <DIV><FONT face="Courier New">3. Then first strand cDNA synthesis is performed
> by polyT17 primer.</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT
> face="Courier New">AACGTGGCTCGAGTGGCTGAACGCTTTTTTTTTTTTTTTTTVN</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">or </FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT
> face="Courier New">AACGTGGCTCGAGTGGCTGAACGCTTTTTTTTTTTTTTTTTV</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">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.</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">To my surprise, after sequencing, I get a little
> of insert (8-80 bp) and long TTTTT afterwards (50 - 250 bp).</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">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.</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">Where does come these long polyT from? Any
> suggestions?</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New">Thank you!</FONT></DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV>
> <DIV><FONT face="Courier New"></FONT>&nbsp;</DIV></BODY></HTML>
> 
> ------=_NextPart_000_0008_01C6FAAC.7213FFB0--



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