Difficulty Sequencing Using Internal Primers

Christine Ward CVMCKW at VTVM1.CC.VT.EDU
Tue Apr 12 17:35:19 EST 1994


Greetings Netter,
   I'm posting this for a friend who has temporarily lost his e-mail:

   I have recently clones and sequenced a gene from Bacteroides fragilis in
 pUC18/DH5a.  I would like now to look upstream and downstream from this gene.
 I find that I can use exact primers (24-mers) from the gene in PCR to isolate
 portions of the gene, but I cannot use the same primers to sequence with.
 Sequencing with these internal primers produces a very faint sequence that is
 impossible to read even when exposure time is lengthened extensively.  Has
 anyone else experienced similar problems and how did you overcome them?? Any
 help would be greatly appreciated.  Thanks in advance.
                                            James Kling
                                            Dept. Biochem. and Anaerobic Micro.
                                            Virginia Tech
P.S. Two notes that may be of interest:
  1.  The gene appears to be toxic to E. coli (i.e. it has been hell to clone)
  2.  Sequncing the ends of the insert with pUC forward and reverse primers
      produces easily readable sequence.

******************************************************************************
Please feel to respond to the net or me directly and I will forward all info
received to J. Kling.  Thanks!

Christine Ward
Virginia Tech
cvmckw at vtvm1.cc.vt.edu



More information about the Methods mailing list