Discussion of DNA Sequence

Darren A. Natale dnatale at box-d.nih.gov
Thu Jul 10 08:57:54 EST 1997


ma wrote:
> 
> Dear Netters:
> 
> I have met many problems in DNA sequence recently, so I like to initiate
> a discuss on this topics. I cloned a PCR product and insert into a
> T-vector. The enzyme check of polylinker is OK. The sequence from one
> end (T7) by DNA automator is OK. But the sequence from another end
> (SP6)did not get  any signal. The sequence was conducted by a commercial
> company since our lab has no such equipment. Could any one give your
> idea what is wrong about it? I also like to know the general
> troublesomes in DNA automation sequence and how to avoid them? Any
> seggestion and discussion is greatly welcome.

Are you using Invitrogen's pCRII vector?  You may think that you are,
but you
actually might be using a different plasmid (pCR2.1).  This second
vector lacks
the SP6 promoter.  Check the tube containing the T-vector to make sure
it is not pCR2.1.  Note that you can still use the M13 forward and
reverse primers with 
both vectors.

D. Natale
dnatale at box-d.nih.gov

Disclaimer:  The view or information given above originated in my own
brain, and 
does not reflect the views of the discorporate entity known as NIH or
any other branch
of the US government.



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