Degenerate primers for direct fragment sequencing

bernie b at ber.com
Thu Jun 2 04:25:54 EST 2005


We use highly degenerate primers (exclusively) and order our primers
with M13 or T7 tails on the 5' end.  After the initial PCR, we then
use M13 or T7 primers for sequencing.

Jerry Regier has also recently published a paper that mentions the
primer design method.  You can find the PDF at:

http://www.umbi.umd.edu/users/jcrlab/PCR_primers.pdf


bernie ball


On 1 Jun 2005 14:57:41 -0700, "amerotto" <amerotto at yahoo.com> wrote:

>I am sequencing fragments from Cyperus difformis (Plant, cyperaceae). I
>am using
>degenarate primers in order to isolate the fragments that I want. For
>some
>primers/template combination I had good results, but for another the
>sequence just not
>appear or appear with double peaks. I am using the Qiagen gel cleaning
>kit and the
>Sigma spin column for the cleaning procedures.
>
>Question: Are degenerate primers a problem for sequencing? If yes, how
>can I compensate
>for that?
>
>Thanks
>
>Aldo
>
>
>Aldo Merotto




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