pCAT Basic cut by Hind III

[Martin Kennedy]

In article <CrKC86.3FD at acsu.buffalo.edu>, vp23qw83 at ubvms.cc.buffalo.edu (Xin Guo) writes:
> I cut pCAT Basic (~4.4kb) using Hind III (one Hind III site) and
> got three bands on agarose gel. Compared with lamda DNA/Hind III
> marker, I saw one the 4.4kb cut linear pCAT band and non-cut
> circular pCAT band. But I also saw a band about 8.8kb. The three
> bands seem to have the same amount of DNA. I am new on molecular
> cloning. I have no idea what the 8.8kb band is. Is it possible
> that the band is ligated two linear 4.4kb DNA or something else?
> I isolated the pCAT from E. coli using phenol/choloroform method.
> 
> Xin Guo

Many plasmids can exist quite stably as catenates of 2 or more molecules; 
sometimes in uncut preps of plasmid you can see dimeric and trimeric forms; 
when completely digested, these will all give a monomeric-sized linear 
species.  They may form by ligation of 2 molecules during construction, or 
by rec-A+ dependent reciprocal recombination.

Different plasmid preps, and different plasmids, show differing degrees 
of this effect, - it is well known in the plasmid literature.  Most native 
(ie non-engineered) plasmids have site-specific recombination systems that 
resolve these multimeric species, but the cloning vectors we use generally 
no longer have this capability. 

See refs:	

Hobom and Hogness (1974) J.Mol.Biol. 88, 65-87
Bedbrook and Ausubel (1976) Cell 9, 707-716
Potter and Dressler (1977) 74, 4168-4172
-- 
Cheers,

Martin

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