aiyar at ebv.oncology.wisc.edu
Thu Sep 12 09:20:11 EST 1996
On 12 Sep 1996 11:46:10 GMT, John Dixon <jpcd0 at mole.bio.cam.ac.uk> wrote:
>If they are, it finally explains to me why this ligation has defeated me
>for so long: the few times that I had left the plates long enough for
>these colonies to grow, I would not have had IPTG/Xgal on the plates and
>then would have assumed that they were satellites.
>Anyone else seen this sort of thing or got tips on how to grow bugs
>unhappy with their plasmid?
I have observed something very similar. A year ago I was
cloning a 1 kb insert into PiVX. PiVX is a small plasmid that
encodes a supF tRNA, but has no antibiotic resistance marker).
The polylinker in PiVX is immediately after the 3' end of the supF
I usually propagate PiVX in MC1061/P3 which contains Kan-wt,
Amp-amber, and Tet-amber genes on a single copy episome (P3).
The selection conditions that I used were Amp - 30 ug/ml,
Tet - 10 ug/ml, and Kan - 15 ug/ml
After ligation and transformation I typically got very few colonies
-- all of which were rearranged forms of the vector alone. Running
the same ligations on gels indicated that the majority of the insert
and vector appeared to be ligated.
In the course of two weeks I screened over 300 colonies, not a single
one of which was positive.
I decided to abandon the approach, but left the last set of
LB/Amp/Tet/Kan plates on my bench for a couple days. To my
surprise a large number of *small* colonies appeared after
about 36 - 48 hours. I picked several of these and they were
all the clone I wanted.
My best explanation is that perhaps insertion of a large insert
3' to the supF tRNA gene interfered with transcription or processing
of the supF transcript, leading to decreased expression of the
Amp and Tet resistance markers.
My solution has been to use significantly less stringent conditions
while transforming ligations of inserts cloned into PiVX.
Ashok Aiyar, Ph.D.
Department of Oncology email: aiyar at ebv.oncology.wisc.edu
University of Wisconsin-Madison tel: (608) 262-6697
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