Predict Possible Recombinants

Note that you have done some of these steps in assignment 4.
Our final screening step will be to determine the nature of the recombinants using restriction enzyme mapping.

In order to decide on a strategy for restriction enzyme mapping, we need to first determine what recombinants we expect to isolate.

To predict possible recombinants we start by looking at the restriction maps for pKC7 and pUC18.

For each plasmid, diagram the fragments you expect to see when the plasmids are digested with BamH I and Hind III (include relevant DNA elements, restriction sites, and fragment sizes).

Any DNA fragments that have compatible ends (i.e. overhanging sequences are complementary – or blunt ends) can ligate together. However, not all ligation products can be replicated by the bacteria’s replication system. What two important characteristics must be present for a recombinant DNA molecule to be replicated?

Diagram all possible, two-piece recombinants (three-piece recombinants are possible but unlikely) that can be replicated. For each recombinant indicate:

  • what plasmid fragments make up each recombinant (the name of the source fragment and the size of the fragment)
  • all relevant plasmid features.
  • list the fragments sizes that will be produced if the recombinant is digested with either BamH I, Hind III, or with both enzymes at the same time.
  • the colour the E. coli colony containing the recombinant will be (when the E. coli is plated on Xgal containing media).

Other considerations
Why don't we expect the larger pKC7 or pUC18 plasmids to self-ligate? (Self-ligation is when a single fragment ligates to itself to form a closed circle.)

In order to clearly identify the recombinant found in each clone, what restriction digest(s) would you need to perform? Keep in mind that you will not be able to ‘see’ the 30 bp fragment on your gel.

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