help with Southern and PCR expts. in lab course

JIM LISSEMORE JLISSEMORE at JCVAXA.JCU.EDU
Wed Sep 10 16:55:43 EST 1997


     This is a request for assistance in improving
Southern blot and PCR exercises that I am currently
using in my molecular genetics lab course.  My
motivation is mostly selfish but I expect that other C.
elegans workers teaching molecular genetics lab courses
will also be interested.
     The basic scenario I have in mind is to isolate
genomic DNA from N2 and from a mutant that contains an
insertion or deletion of about 200-500 bp and that can
be easily detected as an RFLP on a Southern and as
different size PCR products.  In thinking about this
exercise, I have come up with a list of criteria that
need to be met:

--the Southern probe will detect 2-5 restriction
fragments (using six-base cutters) of a single-copy
gene and will be in a plasmid vector

--the complete sequence of the insert and the vector
must be available

--either a cDNA or genomic clone should be fine 

--the complete sequence of the genomic region detected
by the probe must be available so that restriction
fragments detected on the genomic Southern can be
predicted.

--the precise molecular basis of the polymorphism is
known (i.e. which bases have been added to or deleted
from the wildtype gene)

--PCR primers will flank the site of insertion or
deletion and will generate products between ~300-1000
bp

--primers must work reliably (I don't need any more
experience with primers that work only on certain days
of the week) and give good yield.

     If you are working with or know of a gene that
meets these criteria or if you have a gene that would
work with either the Southern or the PCR exercise,
please let me know.  I will gladly post a summary of
responses.  

Thanks very much!

--Jim



Jim Lissemore
Biology Department
John Carroll University
University Heights, OH 44118
email: jlissemore at jcvaxa.jcu.edu
Phone: (216)397-4196
FAX: (216)397-4482



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