Help with Southern Blot Analysis

Chris Hebertson cfh at fullnet.net
Fri Apr 30 18:15:10 EST 1999


A small group of us have been working on a Southern Blot the past few 
weeks in a Molecular Biology course, and there are a few questions
that we had that I thought I would try and post. If you have any
responses, could you please e-mail them to me at
hebertso at cherokee.nsuok.edu

Thanks in advance!

We previously prepared pUC19 plasmids with a lambda DNA insert, and we
used a Southern Blot to determine where in the lambda genome the
insert came from. We cut lambda and ran gels with four different
restriction enzymes - BamH I, EcoR I, Hind III, and PST I. We then
probed the cut lambda with the recombinant pUC19 plasmids.

Upon analysis of the blot we determined that the insert seems to have
come from two different sections of the lambda genome - one in the
5,000-15,000 np range and one in the 35,000-40,000 np range. Is this
possible?

If it is, our assumption is that the insert was constructed by joining
the two different fragments to make one insert - or that there where
actually two different recombinant pUC19 plasmids made. How would you
go about determining which of the two hypotheses are/were correct?

Thanks for your feedback and/or help!! Again, the e-mail adress to
send comments to is - hebertso at cherokee.nsuok.edu

--

Christopher Hebertson
http://fly.to/cfh
"Seriously baby, I can prescribe anything I want!"
- Dr. Nick, from "The Simpsons"




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