SEQUENCING Large Plasmids

Steve Marsh marshs at
Thu Aug 31 13:37:23 EST 1995

In article
<Pine.OSF.3.90.950831155117.11395A-100000 at>,
wylie at CENTRAL.MURDOCH.EDU.AU (Steve Wylie) wrote:

> Has anyone tried to DNA cloned into plasmids of around 12kb? Presumably 
> one would need to add more template than with a smaller plasmid as the 
> proportion of target DNA to the rest of the plasmid is less. ABI tells me 
> it is better not to attempt it, but I would like to check the site of 
> insertion into the vector. Any suggestions most welcome.
> Steve Wylie
> Murdoch University
> Western Australia

We have attepted to sequence 12kb plasmids in our core facility in the
past with mixed results. The chemistry is optimized for a plasmid about
5kb. We had the person bring us template at 2X concentration recommended
by ABI protocols. We had better results with the dye terminator chemistry
that with the dye primer chemistry. The template must be very clean for
this to work. Also longer primers seemed to help (ie about 24-30 bp in
length). You can try but I won't guarantee that it will work or work twice
if it does.
Steve Marsh DNA Sequencing Core Facility @ Caltech.

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