pheacock at utmmg.med.uth.tmc.edu
Tue Nov 15 12:32:23 EST 1994
In article <tan-1511941534150001 at smac.ethz.ch>, tan at mol.biol.ethz.ch (Song
> In article <9411142223.AA04169 at calvin.jci.tju.edu>,
> P_Norton at CALVIN.JCI.TJU.EDU (Dr. Pamela Norton) wrote:
> > As the subject line says, we have been having terrible problems
> > getting a SmaI fragment re-subcloned; essentially we are trying to turn a
> > Sma fragment around. First attempt: cut plasmid with Sma, isolate both
> > bands, ligate back together, look for inserts in the right orientation.
> > Only one or two - but these don't cut with Sma, and sequencing says that a
> > single base is missing at both sites. Start again, with XmaI this time. Got
> > about four in the right orientation, these now cut only once with Sma or
> > Xma. Sequencing reveals that one of the sites (the same in all) has a base
> > missing. The cursed site is within a very GC-rich region, perhaps this is
> > the problem?
> > I am literally tearing my hair out over this one. Does anyone know
> > whether Sma tends to nibble 5' or 3' ends? In other words, would it help if
> > we try to refill any nibbled ends before ligation? I'm thinking of
> > mutagenizing the site back into the plasmid with a single site present
> > (both are in coding sequence, so deletions are a major problem). Before I
> > resort to that, I thought that I would draw on the accumulated net wisdom.
> > All suggestions and relevant anecdotes are welcome.
> Others on the net have reported problems using SmaI digested vector to
> subclone blunt PCR product, and the consensus seems to be that SmaI does
> have an exonuclease problem/contamination. EcoRV appears to be the enzyme
> of choice for subcloning blunt ends, though this won't be applicable in
> your case. Afraid that I don't know exactly what the exonuclease activity
> of SmaI is like (i.e. nibbling 5' vs 3' ends).
> You could try filling in the SmaI ends before ligation as you mentioned.
> Shame that using XmaI didn't help. One would have thought that should
> have worked very well, given the nice sticky ends. I do note that Biolabs
> suggests long incubation times (o/n) for complete cutting of plasmid DNA
> by XmaI. Such long incubation times might aggravate slight exonuclease
> contaminations, of course.
> My only new idea to this is to use AvaI (cuts C/PyCGPuG) instead of SmaI
> or XmaI. I believe it's a relatively good enzyme (it was a long time ago
> since I last used AvaI. perhaps someone else with more extensive or more
> recent experience could post any good or bad comments about AvaI). It's
> relatively inexpensive, at least. The one obvious drawback is that AvaI
> will recognize more sites than SmaI or XmaI because of its degeneracy. So
> I hope that you don't have any other AvaI sites in your plasmid.
> Good luck!!
> Song Tan
> Institute for Molecular Biology and Biophysics
> ETH-Honggerberg (Swiss Federal Institute of Technology)
> 8093 Zurich, Switzerland
> email: tan at mol.biol.ethz.ch
Even better, buy the enzyme PspA I from Stratagene. It is an Isoschizomer of
Xma I that works well. Ihave used it many time with good sucess.
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