Do you need supercoiled pDNA for ds-sequencing?

Roger Wiegand rcwieg at ccmail.monsanto.com
Tue Nov 1 17:57:26 EST 1994


In article <MURIANAP.30.000C1D5C at FOODSCI.PURDUE.EDU>,
MURIANAP at FOODSCI.PURDUE.EDU (Pete Muriana) wrote:

> I recently spoke to a person with a DNA sequencing facility about isolating a 
> plasmid restriction fragment to sequence from without the need to clone the 
> fragment (although the cloning of a plasmid fragment is a simple task). 
Since 
> we have mega-quantities of the plasmid, we thought of gel-eluting the desired 
> fragment; we have a gene-based probe encoded on the fragment (already 
> demonstrated by Southern blot).
> 
> Now the question - the DNA sequencing facility representative indicated that 
> we need to have **supercoiled** plasmid DNA to sequence from.  Is this true? 
> and why wouldn't a linear double-stranded fragment work? Am I missing 
> something here?
> 

This is nonsense. Both cycle sequencing and Sequenase work fine from
linear DNA such as PCR products. We have a number of examples where
nicking or linearizing (either mechanically or using enzymes) plasmids
results in a 5-10X *increase* in signal strength for cycle sequencing.

-- 
Thanks,
Roger

mailto::rcwieg at ccmail.monsanto.com



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