Dear Dr. Blacklock,

Difficult ligations:

1.  If you are working with restriction fragments isolated from an
ethidium-bromide-stained gel, consider switching to GelGreen and a blue
transilluminator; the ligation efficiencies are much higher.  If you don't
have a blue transilluminator, a dissecting microscope set up for GFP
fluorescence is an excellent substitute.  Gels should be post-, not
pre-stained with GelGreen, as prestaining can lead to electrophoretic
artefacts.

2.  By far the best ligation efficiencies are obtained without fragment
isolation of any sort, by simply combining the heat-inactivated
restriction digests and ligating.  You will get all possible combinations
of the restriction fragments, but if you do 24 minipreps there is a good
chance the vector you want will be among them.  Your chances can be
further improved if appropriate restriction sites are available.  Look for
sites which are present in the starting vectors and/or the undesired
ligation products, but not in the vector you wish to obtain.  Cut the
ligation product with such an enzyme (1 hour will do) before transforming
into bacteria.  Enzymes which produce blunt ends or 3' overhangs work
better here than enzymes which produce 5' overhangs; this is
presumably because the coli ligase can religate at the latter sites.


3.  There are some vectors which simply cannot be constucted in E coli, no
matter how many times you try.  You can be fairly sure you have such a
case if a shotgun ligation (point 2, above) produces all the other
expected products, but not the vector you want.  In such cases, consider
assembling your cassette (tet regulated promoter, hairpin, terminator) as
a PCR fragment by PCR-overlapping ends.  Add appropriate fragments of the
DRE transposable element up- and downstream of your cassette, and
cotransform the amplified PCR fragment into tetON-M2 repressor-expressing
cells together with an empty BsR vector.  Select with blasticidin and
screen transformants by PCR, using primers which bind to the tet promoter
and transcription terminator.  With a bit of luck your PCR fragment will
integrate homologously via the terminal DRE fragments into one of the
several hundred copies of the DRE, and the tet-promoter/coding
region/terminator portion will be intact.

General considerations, tet-dependent expression.

Use of vectors like MB38 gives hundred- to thousand-fold variation in
expression intensity; this has been adequately documented in the
vanHaastert group publications.  This seems likely to lead to a variable
phenotype.  You can get around this problem by targetting expression
cassettes to the DRE; this can be done by both PCR- and vector-based
strategies.  In cells transformed with a single-copy tetP-GFP vector
targetted to the DRE, tet-dependent fluorescence is readily measurable in
the flow cytometer, and almost all cells show expression levels within a
threefold range.


I hope this helps,

Harry MacWilliams

----------------------------------------------

On Wed, 19 May 2010, Brenda Blacklock wrote:

> Hello All,
>
> We are trying to construct a long hairpin for RNAi using essentially the
> technique described by Rosel and Kimmel for COP9.  We have the long arm
> of the hairpin (1100 bp) in pMB38 but are having problems inserting the
> short arm (800 bp).  Does anyone have any tricks for getting what seems
> to be a difficult ligation to work?
>
> Thanks for any suggestions you may have for us.
>
> Brenda Blacklock
>

----------------------------

Prof. Harry K. MacWilliams
Biologiezentrum der Ludwig-Maximilians-Universität
Grosshadenerstrasse 2
82152 Planegg-Martinsried
GERMANY

tel (office) +89 2180 74 288
tel (lab)    +89 2180 74 289
fax.         +89 2180 74 219

callers from North America please dial:
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