integrative plasmids

FATIMA at AIMP.UNA.AC.AT FATIMA at AIMP.UNA.AC.AT
Thu Jul 8 04:16:00 EST 1993


(Darin - obrien at molbio.cbs.umn.edu asking whether integration of a 22kb
plasmid is likely to work)

I do not have any experience with such a large integrative construct;
however, a 10 kb YIplac128 derivative worked fine. Anyway, I would try
to cut down the construct size, if possible, already because of the
bacterial manipulation; also, I can imagine that the larger the piece 
of DNA is, the more likely it can recombine with more than 1 site in
the yeast genome - or it could even have an intrinsic ARS activity,
which is a little disaster when you try to integrate it.
A general note on integrative transformations: most versions of the TRP1
marker on plasmids have a truncated promoter and are poorly expressed
at single copy, so that wrong transformants (i.e. convertants in the
TRP1 locus) are strongly selected for. And, when making an integrative 
construct yourself, avoid using the TRP1 EcoRI-EcoRI fragment!!! It has
a strong ARS; even if you just transform this fragment as linear DNA
into yeast, you will end up with a nice little high copy number
plasmid. After some bad experience, I am now avoiding TRP1 integrative
transformations unless really necessary.

Good luck!              Fatima (fatima at aimp.una.ac.at)



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