Blunt-end cloning

Dom Spinella dspinella at chugaibio.com
Fri Aug 15 17:15:38 EST 1997


Mikhail Alexeyev writes (in response to the previous posting):
> In some of my clonings of short (up to 600 bp that is) PCR products
> Recombinant clones had a light blue color. This happened only in
> particular insert orientation.
> 
> On the other hand, I hope you did not phosphorylate your primers,
> because if you did, then there is a good chance for insert
> multimerization (high insert to vector ratio)which would mess up
> everything.
> 
> M.A.

I must disagree with the last statement.  Attempting to blunt clone a
non-phosphorylated insert (PCR-generated or otherwise) into a
phosphorylated blunt-ended vector will lead almost exclusively to
re-ligation of the vector. The intra-molecular ligation reaction will be
highly favored. Of course, the re-ligated vector will generate blue
colonies, and one could argue that one merely need select the white
colonies.  However, these are usually pretty rare -- almost
approximating the frequency of the mutation rate in the LacZ gene,
especially with blunt ligations.  As the previous posting observes, it
is not uncommon for white colonies to be formed by bugs with plasmids
having no insert. As for generation of "multimers" of insert, again this
can occur but will be a relatively rare event.  Whether with blunt or
sticky-end cloning, is has been the standard for years to clone a
phosphorylated insert into a dephosphorylatred vector and I have seen no
data to contradict the conventional wisdom. Moreover, insert multimers,
even if they occur (and they're virtually always dimers if they do
occur), don't "mess up everything".  Clearly it is not the problem
described in the original posting in which there was no insert at all
when attempting to blunt clone a PCR product generated by Pfu.  I'd be
interested in hearing other opinions.  However, in my experience a
common mistake made by the inexperienced in blunt-cloning a PCR product
is to use a blunt-ended, dephosphorylated cloning vector, but forget to
phosphorylate the primers or the PCR product prior to ligation. 
Obviously in that case,   no phosphate = no ligation.  -- D.G. Spinella



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