Plasmid sequencing: Methods to improve resolution

[Jeff Lawrence]

In article <S941407.1.00174A94 at mailserv.cuhk.hk>, S941407 at mailserv.cuhk.hk
wrote:

> I would like to know how I can get better-resolution sequencing (bands 
> towards the end of the gel are hardly distinguished). Moreover, are there any 
> methods to increase the number of sequence obtained each time(other than 
> buffer-gradient page)?
> Frankie, T. C. Lee

i extend reads by (1) using the sequenase extending mixes and (2) making
the plasmid single stranded.  to do this, i typically cut with an
appropriate endonuclease, phosphatase, and cut with another 'zyme to yield
a 5' phosphorylated end.  the linased end can be digested with the lambda
exonucleoase, leaving the phosphatase-ended strand intact.  ssDNA reads
*much* further than dsDNA.  
jeff

-- 
jeff lawrence
lawrence at bioscience.utah.edu