Blunt-end cloning

Dr. Jeney Csaba jencsa at net.sote.hu
Thu Aug 21 10:27:30 EST 1997


dspinella at chugaibio.com (Dom Spinella) wrote:

>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

Partly..partly...I agree partly with both of them...first of all it
should be emphasized that the concentrations of the vector and the
insert together drive the ligation reaction and so the
multimerization, which in turn can 'mess up everything'...
to shed some light on this: in practice the not too frequent encounter
with dimerization is really the rule but it do not mean that larger
multimers are infrequent, bacause if multimerization occurs the longer
DNA strands will favor further linear growth and the these lienar
giants do not transform well...to put a huge amount of DNA in the
ligation reaction is the other general error and will display this
situation and result in almost no colonies (in the case in question it
seems to be not this is the problem)...so anyway check out the
concentrations of both of the vector and the insert

The phosphorilation is recommended because the ligase mostly dislike
such half-phosphorilated templates biassing the reaction toward the
vector circularization.

One more point: try another DNA isolation method...in my experience
most of the foolish cases could be tracked down to the black horses of
these methods....
....otherwise the science is the place where everything should not be
fine.

Anyway good luck

Csaba





More information about the Methods mailing list