cloning woes

Robot-Boy s535290 at aix1.uottawa.ca
Wed Jan 28 13:56:12 EST 1998


Hi all,

I'm having a little bit of a problem cloning this particular fragment into
my expression vector of choice. It is an NcoI/HindIII fragment of 1.7
Kb. The fragment is obtained by a lengthy process of partial digestion/gel
purification/digestion with a second enzyme/gel purification. I am using
guanosine on the purification gels and trying to minimize UV exposure. The
problem is that after all the manipulations, the yield of my fragment of
interest is pretty minuscule. Nonetheless, it should be clonable. My
vector (a yeast shuttle vector of approximately 10 Kb minus insert) is cut
with NcoI and HindIII to accept the fragment, and though theoretically not
required, I am phosphatasing and gel purifying it as well. I can put an
NcoI/HindIII fragment into this vector as a positive control. Mind you, I
have oodles of the test insert, and little of my "holy" fragment. My
ligation controls are working, the vector ligates to test inserts
just fine but what I am worried about is transformation efficiency.
The efficiency of my cells (done using the TSS protocol) is 10^7 using ccc
pUC18, but this plasmid is much larger. I have tried this cloning a number
of times, and I honestly think that what is holding me back is the
combination of low yield of the clonable fragment of interest and the fact
that the vector is so large and my cells are not ultra competent. What do
you guys think ?

fwiw, an internal NcoI site is the real problem. It cuts my coding
sequence right near to the HindIII site :

		N---------------K----------------N-H
		|--------------1.7Kb---------------|

I have triend blunting the NcoI site and three way cloning using a KpnI
site near the middle of the coding sequence, and neither of these
approaches have worked. I just figured that as long as I could generate
enough of the full length NcoI/HindIII fragment through partial digestion,
this *should* be clonable but no such luck yet.

any ideas ? TIA,

Ed




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