Discussion of DNA Sequence

Eric Anderson e-anderson at ski.mskcc.org
Thu Jul 10 08:37:35 EST 1997


In article <33C55CA1.3E9E at ns.east.cn.net>, ddsyr at ns.east.cn.net 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.
>
>Dr Q. H. Ma
>Institute of Botany, Academia Sinica
>Beijing 100093, China

the problem is probably the SP6 primer.  it doesn't work very well under
cycle sequencing conditions and in 14 months of running a small automated
sequencing facility i never once saw a good sequence obtained from SP6
primer.  is there another primer site at that end of the vector (i.e. T3
or an M13)?  either one of those will work nicely.

good luck,

eric

-- 
Eric C. Anderson
Sloan-Kettering Institute for Cancer Research 
Dept. of Cell Biology and Genetics
Lab: (212) 639-2977
Fax: (212) 717-3298
e-anderson at ski.mskcc.org



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