Do you need supercoiled pDNA for ds-sequencing?

Jim Owens jow at helix.nih.gov
Tue Nov 1 17:11:40 EST 1994


In article <MURIANAP.30.000C1D5C at FOODSCI.PURDUE.EDU> Pete Muriana,
MURIANAP at FOODSCI.PURDUE.EDU writes:
>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?

I can only speculate.  My experience agrees with your sequencing facility
representative.  I have always had better luck sequencing intact, mostly
supercoiled plasmid than with linear double-stranded DNA.

Maybe there is less likelyhood of reannealing of the strands when coiled
DNAs are snap denatured?

Good luck,

Jim Owens



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