Three fragment ligation
gilles at p450bm3.swmed.edu
Thu Jan 23 14:09:48 EST 1997
Andrew Doherty wrote:
> Hi everyone
> There must be lots of people out there ligating three bits of DNA
> together instead of just two - but I'm having major problems with it.
> I'm constructing a fusion protein, but have very few restriction sites
> to choose from which I can use, hence the need for the three fragments.
> My vector is pcDNA1/amp (dephosphroylated) cut with NotI/SphI, my
> receptor cut with NotI/HindIII and GFP cut with HindIII/SphI. So putting
> all three together should give me a circular plasmid. The ligation looks
> fine on a gel - the GFP (800bp) and receptor (3Kb) both disappear and I
> get a ladder of products. But no colonies!! I know that the bugs are
> fine, as other transformations are working well - so my question is, are
> there any tricks in getting this sort of ligation to work or is it just
> luck??!!@# I'm using BM's T4 ligase at 4 deg C overnight.
Would it just simply be that the expression of your fusion is toxic? I
personnally don't see any reason why it should not work, specially if
the ligation appears to happen, on a gel. Maybe you should try different
strains of E coli, just to be sure. And if possible try a LacIq strain,
that will repress very well the expression of betagal, and of your
protein to presumably.
Another thing that could have happen. If the two sites on the vector
are close enough, you might not distinguish between a single cut vector
(either by Not I or Sph I) and a double cut vector. As you
dephosphorylate the DNA, if you have only one cut, then you cannot do
the third part ligation and cannot religate the vector on itself. To
check that, you should be sure your enzyme actually cut you DNA, when
they are alone (and hope they do so when they are together!!!!)
Good luck anyway, as it seems that never hurt in Molecular Biology!!!!!
Dr. Gilles TRUAN Phone # :(214) 648-4741
Dpt of Biochemistry Fax # :(214) 648-8856
UTSW Medical Center E-mail :truan at utsw.swmed.edu
5323 Harry Hines Blvd
Dallas, TX, 75235-9038
Tout va très bien, Madame la Marquise,
tout va très bien, tout va très bien...
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