How to seq. a single-primer PCR product

Hiranya Roychowdhury hroychow at NMSU.EDU
Thu Jul 2 15:13:16 EST 1998


At 03:57 PM 7/2/98 GMT, Brian Sydnor Burnes wrote:
>Hello fellow labrats!  I have PCR products generated from inverse PCR
>primed off the inverted repeats of a Tn5 insertion.  Only one primer was
>used, so the ends of the products are inverted repeats like the
>transposon.  How can I directly sequence these products without getting
>overlapping forward and reverse reads?  Sequence an asymetric digest?

If you have an idea of the RE map of your amplicon(s), this is a good way to
go. Reactions for automated sequencers can employ differentially labelled
primers where the reactions carried out in the same tube would be readable
from both ends. In such cases, RE digests are not necessary, and, for a
reasonably sized template, you may actually read overlaping sequence through
the middle of the template - thereby confirming that portion of the
sequence. For a very large number of amplicons, direct sequencing with the
same primers would be desirable, otherwise,

>Ligate (blunt) into a sequencing vector?  

this may be the better way since you'll have a larger supply of template for
future. T/A cloning, using a vector like pGEMT, works more efficiently than
blunt-end cloning. 

I appreciate any input; thanks.
>
>
>--
>Brian Sydnor Burnes
>Georgia Institute of Technology, Atlanta Georgia, 30332
>uucp:	  ...!{decvax,hplabs,ncar,purdue,rutgers}!gatech!prism!gt2837a
>Internet: gt2837a at prism.gatech.edu
>
>


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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