PC at no.email.sorry
Wed Feb 18 19:23:42 EST 2004
that happens only with this pair of primers?
I had it once - a nightmare, four weeks were spent: PCR was just great,
but nothing further worked. At the end I gave up on the restriction and
tried to clone the fragment into a Topo vector - even that did not work
well. I finally got a couple clones, each of them had a part of the good
sequence (same thing - deletions at the ends) - from these two I
assambled the complete cDNA. After that all other primers on the same
cDNA worked well, it was re-cloned by now into at least a dozen vectors...
my explanation - something was wrong with the oligos - not completely
deprotected may be? Funny thing is that the ligation worked - I could
see the ladder of the insert self-ligation. As if the DNA did not
replicate in E. coli.... Deprotection is simple (but stinky) - just
heat the oligo in ammonia for a while - you probbably know it better, as
you make the oligos yourself.
Paul Shinn wrote:
> We have run into some problems with our ligations. Our transformants
> are either non-existent, non-abundant, incorrectly cloned, or vector
> We do directional cloning of PCR products using Sfi I and all our
> transformants are sequence verified by us. The vector has 2 Sfi sites
> and is digested with Sfi I and then gel purified. Because of how Sfi
> cuts, there should be no self ligation of the vector and the PCR products
> (ends are also digested with Sfi) should clone in only one direction.
> GGCCN NNN|NGGCC
> CCGGN|NNN NCCGG
> But like I said, we are getting vector only clones or the transformants
> have backward inserts or there is truncation of the PCR product ends
> during the ligation. And sometimes (rarely) they actually work. Most of
> the clones look like they are blunt end ligated rather than sticky end
> because the Sfi site is gone. Sometimes the Sfi is gone on the insert
> side and sometimes on the vector side. There is no rhyme or reason to
> For the most part the PCR is using Taq. Then sodium acetate/ethanol
> precipitated. Digested with Sfi overnight. Precipitated again and then
> ligated overnight at 16. For larger inserts we use a proof reader and
> then do phenol/chloroform and then sodium acetate precip. We had some
> problems with the proof reader chewing back the ends of the insert.
> I synthesize the PCR primers for cloning. The forward primer is 56mer;
> the reverse primer is 43mer. If the oligos are degraded, the 3' end would
> usually go first, right? If so, the restriction sites at the 5' end
> should be intact. PCRs are still working, too.
> Each person has their own stock of vector and generates their own clones.
> It's hard to believe we're all doing something wrong at the same time.
> I worry that we're seeing the result of multiple problems like vector is
> degraded and PCR ends are degraded independently. Can a sticky end blunt
> ligate to a blunt end?
> We're out of ideas. Thanks for your help.
More information about the Methods