cloning blunt

Steve Hartson shartson at BMB-FS1.BIOCHEM.OKSTATE.EDU
Wed Nov 15 14:05:07 EST 1995


Nandy Hofland (nandy at ruly46.medfac.leidenuniv.nl) wrote:

For two months now I have been trying to clone a 2 kb blunt fragment
(NruI/HindIII, filled in with klenow) into a PvuII site of a 6.5 kb vector.
I am aware of the fact that blunt ligation are not as efficient as sticky
end ligations, but still it should work.
There are some specific questions I would like to put forward:
1. Self ligation of the PvuII cut vector does not work. Is anyone aware of
problems specific for a blunt PvuII site?
2. From the catalog a learned that blunt NruI sites are difficult to
ligate. Does anybody know why, and if there is a way to imporve this?

Any further suggestions on how to improve blunt ligation efficiency are
most welcom!
---------------------
Nandy,

        While I can supply no specifics on 1 or 2, I do have "further
suggestions".  In our lab we use a specialized vector that has only one
site for inserts and no blue/white selection and, thus, we routinely stuff
blunted inserts into this blunted site (BglII).  Our experience has been
that blunt-end ligations are so very inefficient (appr. 1/5000th that of
sticky?) that each of the following parameters must be working perfectly:

        (1)  Start with lots and lots of DNA.  Cut appr. 5 microgram (3-5
Kb plasmids) in 0.2 ml digests, phenol extract, and ppt.  Redissolve the
insert in appr. 20 microliters TE.  Dissolve the vector in appr. 10
microliters of water and treat with phosphatase w/ buffer (appr. 1
microliter P'ase in 20 microliter final volume).  Load dissolved insert and
phosphatased vector directly to distal lanes of an agarose gel such that
the desired bands contain 1-3 micrograms of DNA.  Note that bands will be
ugly, but you're not interested in pretty, you need concentration.
        Excise just the center of the band, extract, and ligate as per Jim
Graham's excellent protocol archived in 9304: "Consistent subcloning from
agarose (finally)"-it works great!!!

        (2)  Quantitate the competency of your competent cells using
supercoiled DNA; I suggest the uncut vector.  In our experience, you need
about 5e8 cfu/microgram DNA or you might as well stay in bed.  I like
Stratagene's XL1blue supercompetents.

        (3)  If your ligase/ligase buffer is old (6 mo), buy new ("time is
money").

        (4)  Ligate at RT for 8+ hr (sometimes old ligations will yield
cfu's following storage in the 'fridge).

        Using these protocols with (2) and (3) working OK, I can usually
get about 50 colonies from 1 microliter of ligation transformed directly
into 20 microliters of cells:  25% "vector", 25% funky rearrangements, 50%
inserts.
For forced-directional sticky-end cloning, I get a pseudo-lawn of
transformants containing insert.

You may also wish to consider an alternative strategy such as
linker/adapter or shuttle-vector modification of your troublesome
restriction sites.

                                'luck,
                                        Steve


///////////////////////////////////////////////////////////////////////

Steve Hartson
Dept. Biochem. and Mol. Biol.
Okla. St. U.
Stillwater, OK  74074  USA
shartson at bmb-fs1.biochem.okstate.edu





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