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DNA sequencing

Corinna. Dutton Corinna.Dutton at newcastle.ac.uk
Fri Dec 8 11:01:45 EST 1995


I have had problems with automated DNA sequencing (Taq method), whereby 
the samples used give no data.  The DNA concentration has been checked 
both using a spectrophotometer and on agarose gels comparing it to 
samples that have sequenced correctly.  A collegue suggested that the 
plasmid (Bluescript) had become deleted at the primer site (forward 
primer), and so this was digested with Kpn 1 and low and behold samples 
that had sequenced cut, and those that didn't , didn't. Sac 1 sites do 
seem to cut and so I'm going to try the standard reverse primer. Has 
anyone else had these problems, as it seems illogical that my 200bp insert 
can be ligated and cut out of the plasmid but it has deletions?

Corinna 



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