Annealing of two complementary circular SS DNAs

Robert Seymour r.seymour at prospect.anprod.csiro.au
Sun Aug 18 18:12:51 EST 1996


In article <graham-1608961305340001 at tastiger.dbe.csiro.au>,
   graham at dbe.csiro.au (Lloyd Graham) wrote:
>A procedure I am conducting will generate two closed 
circular
>single-stranded DNAs which are perfectly complementary to 
each other. I
>would like to anneal them together to generate a 
double-stranded DNA (a
>3.6kb plasmid). Is it simply a matter of heating to 95'C 
and allowing them
>to cool slowly, or will their circular nature complicate 
things?
>
>Ultimately I just want to transform E.coli with the 
resulting plasmid.
>Perhaps it's not even necessary for me to do an annealing 
step - does
>anybody know if transforming with single-stranded plasmid 
DNA is
>effective? 
>
>Many thanks,
>
>Lloyd Graham
>CSIRO Div. Biomol. Eng., Australia

Hi 

 If all you wanted to do was to synthesise a plasmid, I 
would have thought that it would have been easier to 
synthesise one closed circle and then a small primer (15 
mer) that annealed somewhere on that circle.  This primer 
could then be used to prime the synthesis of the second 
strand using DNA polymerase, prior to transformation.  The 
only thing I would be unsure of is whether DNA polymerase 
would generate a second strand around a circle, but its 
probably worth a try

Robert Seymour
CSIRO, Division of Animal Production, Australia



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