PCR alternative to plasmid rescue?
Mark D. Garfinkel
garfinkl at iitmax.iit.edu
Mon Feb 21 14:35:50 EST 1994
gvacano at eagle.wesleyan.edu writes:
> Our lab has been doing P-element transformation for some time now,
>using pCaSpeR vectors (esp. 3 and 4). We've obtained a few transformants that
>are homozygous lethal, and we'd like to obtain sequence information for the
>region(s) in which the P-element cassette(s) are located. [...]
All the components of pCaSpeR have been sequenced, i.e., the
white minigene, polylinker, and P element segments, and so it should
be easy for you to assemble your own sequence file from first principles.
Ashburner's Big Grey Book has a chapter on transformation vectors that
will give the maps & references.
Tricks on how to design the primers & do the inverse PCR can be
found in several papers that discuss cloning single-P insertions from
pools of diverse genotypes *without* knowing the phenotypes in advance.
Dennis Ballinger is one name to search for; Kim Kaiser another. Kaiser &
Kevin O'Hare published a TIG article about these methods 2-3 years ago.
Also Palazzolo's people (key name Bruce Hamilton?) have done similar.
Hope this helps.
Mark D. Garfinkel (e-mail: garfinkl at iitmax.acc.iit.edu)
My views are my own, which is why they're copyright 1994
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