Cloning sticky PCR products (Breakthrough?)

Jim Graham graham at biodec.wustl.edu_nospammmmmmm
Wed Apr 15 04:26:57 EST 1998


Hello Colleauges,

This morning's Biotechniques (24 no.4 p578-580 1998) has what I find a
major insight to a longstanding problem in the lab which has been
discussed ever since the "dawn" of PCR a few years back.

"What's with the clonability of PCR products?"

Certainly the nontemplated addition of A, now admittedly a couple A's,
to about half the PCR product (in my hands) paritally explains the
failure to blunt end ligate efficiently.

However, the fact has remained that even when restriction sites are
engineered into the PCR product so that they are 20 or more nucleotides
from the ends, the subsequent ligation of the sticky-ended product is
quite inefficient. (You may not have noticed it if you are looking for a
only a single clone.) Now Wybranietz and Lauer at Medical University
Clinic Tubingen demonstrate that such sticky-ended fragments derived
from PCR are greatly improved in their ability to concatermize (ligate)
when purified by several high stringecy steps designed to eliminate
contaminants.

The authors show that a Quiaquick PCR column (chaotropic agent and silca
matrix) alone leaves the ligation efficeincy rather depressed, while a
proteinase K digest and phenol extraction is marginally better. However,
a proteinase K treatment, phenol extraction, Quiaquick column,
restriction digest, and another Quiaquick column dramatically imporves
subsequenct sticky-end ligation. To their credit, the authors describe
this as a 5 hour process. :)

I'm thinking, what if one were to heat of the chaotropic binding buffer
prior to cooling and column purification? Would that do the trick alone?
Can Taq renature in "chaotropic" salt used for Quiaquick columns? (Has
anyone tried this?) A friend here has noticed that merely two
Quaquickings, before and after digestion, gave a decent boost in
transformants obtained.

Kudos to Wybranietz and Lauer. Anyone have an alternative approach and
some data?

Best regards,

Jim
J. Graham PhD
Biology Department
(Now buying a kit or two :(

Drop the _nospammmmm to reply.



More information about the Methods mailing list