the problem on plasmid ligation and transformation

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Aug 17 11:42:02 EST 1999


Hi Bill,

Bill_A_Nussbaumer at ms.bd.com wrote:

> Hi,
>
> I have a couple of questions.
>
> 1.  What is a concatemer?
>

a multimer of identical DNA fragments, in either of both possible orientations
(head-to-tail or head-to-head). It is produced by ligating not a mixture of
fragments, but only one fragment. Of course, ends must be compatible, and can
either be sticky or blunt (as in the case of PCR products made by a proofreading
polymerase). Blunts will be a bit harder to ligate but still work very well. On a
gel, concatemers appear as a ladder of bands, with decreasing intensities from the
monomer to the higher multimers. It is not difficult to get 5-7mers in a standard
ligation reaction.

>
> 2.  Is it possible to order PCR primers already 5' phosphorylated and hence not
> need the T4 PNK step?

Yes, it actually works better because all primers have a phosphate, compared to
the efficiency to PNK which is never 100%, of course. I have proposed the PNK step
because I assumed that the original poster already has the primers and wants to
use them rather than ordering new ones. The PNK method works efficiently enough in
my hands to get a good number of clones.

Frank





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