cloning dimers

Andrew Hayhurst a.hayhurst at abdn.ac.uk
Fri Oct 3 08:49:18 EST 1997


It is possible to clone multimers of fragments as head to tail units. 
The bugs such as XL1-Blue and SURE tolerate upto a certain number and
then begin to delete and rearrange things!  My own case was
multimerising the HCMV enhancer borne on a 520bp fragment and I got up
to 8 (within a pUC vector) before problems arose.  However, I ensured
only head to tails were being formed by judicious use of terminal
restriction sites.  What you may have is head to head and tail to tail
(inverted repeats) forming which, if they are over a certain size, are
extremely difficult to stabilise within the common strains.  There was a
Gene paper that employed destruction of a large palindrome as a positive
selection in standard hosts.  They used a special strain to propagate
the unmodified vector.  The paper is by Elhai and Wolk, Gene 68:119-138
(1988)- A class of positive selection vectors based on the non-viability
of palindrome containing plasmids that allows cloning into long
polylinkers.
	Hope this is of use, goodluck.



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