The problem is rather common. It's not you or your cells, it's got
something to do with the PCR process. Any fragment generated by PCR is
difficult to clone for some reason. Things smaller than 500 bp seem to
clone pretty easily, no matter what you do (or don't do). Fragments bigger
than 1000 bp are very difficult. Things in between seem very variable.
Some have tried adding long (greater than 10 bp) extentions beyond
the restriction site in the oligo (with some success). Amersham has a kit
out that's supposed to work, and clontech has another kit that's supposed to
work. Both are very expensive. I have shown that there is something odd
about the ends. I cannot cut restriction sites within 6 bases of the end.
I cannot ligate blunt after Klenow, T4 pol, S-1 nuclease, phenol, Proteinase
K, etc. I can cut internal sites and clone an internal fragment. A friend
of mine and I are working on a cheap way to circumvent these problems. If
it works, we will probably publish. In the meantime, he has his way, and
I have mine, and we're both trying both to see which works better. For
science, I don't care who wins, but some good beer is riding on it for now.
Wish me luck in my upcoming PCR cloning face off, and I'll wish you luck in
finding a way to circumvent the problem yourself.
Leonard N. Bloksberg
PreissJ at clvax1.cl.msu.edu
Dept. of Biochemistry
Michigan State University