Q] Circular DNA size determination

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Fri Jun 19 12:07:11 EST 1998


Hi Hyunmin,
There is no problem to run undigested (i.e. circular) DNA on an agarose gel
and to compare its size with that of another plasmid. Indeed, we routinely
check individual colonies from cloning experiments in this way to find those
with an insert. Remember that supercoiled (formI) DNA runs FASTER in the gel
than the linear formIII so there is no easy way to tell the exact size of the
plasmid. However, that is not necessary if you simply want to find clones with
an insert (or, in your case, a deletion). In fact, a 300bp difference in a 4kb
plasmid is very easy to see, even easier than in the digested form.

So, I recommend you run your undigested miniprep DNA from several clones side
by side with a non-deleted plasmid (undigested, too), and look for those that
are smaller than the original plasmid. Once you found a clone, characterize it
with restriction enzymes (and sequencing).
Usually your approach of deleting a part of the DNA is VERY efficient, so
don´t be surprised to see that all clones are positive...

Hope this helps,
Frank



Hyunmin Kim wrote:
> 
> I have delt with circular Plasmid(4kb).
> It has 2 site for SalI between 300bp distance.
> 
> I digested this plasmid with SalI, and I self ligated this plasmid.
> Then  I will have a plasmid with 300bp deleted(=3.7kb).
> 
> To distinguish whether  a certain plasmid is 3.7kb or 4.0kb, Do I must
> linearize the plasmid with appropriate restriction enzyme?
> 
> If both 3.7kb and 4.0kb circular plasmid is applied to
> electrophoresis(with no Resctiriction enzyme added), There is no way
> to distinguish 3.7, and 4.0kb ?
> 
> I would like to know if there is any method to distinguish 2 different
> size Circular DNAs without linearzing the DNAs(no restricion enzyme
> added.)
> 
> (Because it is uncomfortable, and enzymes cost much.)
> 
> 
> --
> Hyunmin Kim
> Laboratory of Microbiology
> Department of Biology
> College of Natural Science
> Hanyang Univ.
> Haengdang-dong 17                Fax   : 082-02-290-0952
> Seoul, South Korea               E-mail: vurk at hymail.hanyang.ac.kr
>



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