Hi L. v. d. Zande
Yes, I recognize your problems about cloning PCR amplified
DNA fragments. I have encountered several and have read several
possible proposals for solutions.
It seems that anything below 500 bp in length can be blunt
ended into a SmaI site pretty easily. Anything longer than 1000
bp seems hopeless. People have tried T4 pol, Klenow,
Kinase, Proteinase, phenol, etc. and all work on things less than
500 bp (which clone fine anyway) but not on big stuff. If you
have a chance to design your primers, add restriction sites, but
terminal sites don't cut. You must leave at least 6 nt overhang
past the site to get cutting.
I had proposed, but have not gotten back to trying yet,
ligating kinased linkers (at high conc.) to the ends (since small
things seem to work, maybe it goes in reverse too with things
working to small things). Once ligated on, cut and ligate into
an appropriate site.
Try this and let me know how it works please. Good luck.
Dr. Leonard N. Bloksberg
PreissJ at clvax1.cl.msu.edu
Dept. of Biochemistry
Michigan State University