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Lecture 7, part 1 of 3
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September 21, 2017
RECOMBINANT DNA


Learning checklist:

1. Know how to predict the fragments from a restriction digest of a linear or circular fragment, for which the locations of restriction sites are known.
2. Know how to infer the locations of restriction sites, given the sizes of fragments in a restriction digest, as measured by gel electrophoresis.
3. Understand how DNA ligases join restriction fragments with compatible ends.
4. Understand how restriction fragments can be cloned into plasmid vectors.
5. Know that DNA polymerases extend a 3' recessed end of an existing DNA duplex, using the complementary overhang end as a template. Be able to show how this principle can be used to label DNA by extending a primer in the presence of labeled nucleotides.

III. RESTRICTION MAPPING

A. Example of a restriction map and corresponding digests

Given a map of the locations of restriction sites, it is relatively easy to predict the fragments we would see in gel electrophoresis. Make sure you understand why we see the fragments in each of the predicted digests. A negative control (-) is included to show that we expect to see a 1650 bp band if no restriction digestion of the fragment was performed.


B. Inferring a Restriction Map from Single and Double Digests 

For each of the following, see if you can figure out the restriction map.

IV. LIGATION AND CLONING

A. DNA Ligases

    1.Chemistry of ligation

    Note: This will not re-cut with BamH1 or Bgl2, but WILL cut with Sau3A (GATC)
    ANY Sau3A site is compatible with BamH1 or Bgl2. 1/16th of the time, site will be recreated.
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    previous page PLNT3140 Introductory Cytogenetics
    Lecture 7, part 1 of 3
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