Difficulty Sequencing Using Internal Primers

h9290184 at hkucc.hku.hk h9290184 at hkucc.hku.hk
Fri Apr 15 21:09:18 EST 1994


In article <199404122235.PAA11688 at net.bio.net>, CVMCKW at VTVM1.CC.VT.EDU (Christine Ward) writes:
> 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

That's puzzling to me too. You did not mention how the sequencing was done. If
it was cycle-sequencing then I am lost. May be not, assuming that it was cycle
sequencing, maybe there is a difference in buffer. You should try repeating the
successful PCR using the buffer used for sequencing. If the sequencing was by
Sequenase then secondary structure come to mind, the primer cannot anneal to
its target sequence.
Please post reponses or at least the successful remedy(ies) to this problem.

Kaimin Chan
IMB/HKU
HK
h9290184 at hkucc.hku.hk



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