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    Reconstructing a Tn10 phage element

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    When phage λ is grown on a Tn10-containing E. coli host, rare phage that transduce tetracycline-resistance can be isolated. You isolate DNA from λ and from λ-tet and cut each with EcoRI and BamHI and see the following sized fragments on an agarose gel:

    Tn10 is 9kb long, with one EcoRI site 3.5kb from its left end (and there are no BamHI sites).

    Construct a restriction map of λ showing the positions of the EcoRI and BamHI sites and the position and orientation of the Tn10 insertion in λ-tet.

    See attached file for full problem description.

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    Solution Preview

    Let's start by looking at the digests. First the lamba phage is cut with
    -EcoRI to yield 20kb, 13kb, 12kb, 5kb (total is 50kb)
    -BamHI 22kb,14kb, 10kb, 4kb (total 50kb)
    -BamHI and EcoRI to yield 14kb, 13kb, 9kb, 6kb, 4kb, 3kb, 1kb (total is 50kb)

    Reconstructing the plasmid is kind of like putting together a puzzle. Let's start with trying to identify bands:
    -both of the large kb bands are cut in the double digest which means they contain both restriction enzymes
    -the 14kb in the double digest may be the BamHI-14k-BamHI or it might have been a cleavage product of the
    -likewise with the 13kb fragment
    -the 9kb double digest has to be a BamHI-9kb-EcoRI ...

    Solution Summary

    The expert examines reconstructing a Tn1 phage element. The sized fragments on an agarose gel is determined.