Help: Difficult RE digest

Shiao Wang Shiao.Wang at usm.edu
Fri Oct 2 12:47:28 EST 1998


> I am using BamHI as I need to produce a single linear fragment for
> cloning.  I am sure the BamHI site is unique as digestion of my mtDNA
> with EcoRI produces a single fragment, while double digestion with both
> enzymes produces 2 fragments (12kb & 6kb).  When digesting with EcoRI a
> slight band shift (when compared with uncut DNA) is observed.  This band
> shift is not observed upon single digestion with BamHI.  In addition,
> attempts to clone mtDNA digested with BamHI have failed (I have also
> tried to ligate my fragment to lambda arms with no success!)  As the
> BamHI/EcoRI restriction patterns in this species do not vary between
> animals I am convinced the sites are associated with conserved regions
> of the genome.  I am also convinced that digestion with BamHI, first
> requires digestion at a second site (I think the BamHI site may be
> within a secondary structure????)  Does anyone have any suggestions -
> particularly relating to digestion within secondary structures?

Lyndal, if I understand your problem correctly, you would like to clone
the entire mtDNA using BamHI but that BamHI won't digest the DNA unless
EcoRI was present. Why don't you just ligate the double digested DNA
with BamHI lambda arms and then look at clones with the correct sized
insert? The EcoRI ends would ligate to each other and the BamHI ends
would ligate to the arms. Sounds too easy, I may have misunderstood your
question.

-- 
Shiao Y. Wang
Department of Biological Sciences
University of Southern Mississippi



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