Do you need supercoiled pDNA for ds-sequencing?
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).
> 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.
mailto::rcwieg at ccmail.monsanto.com
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