Ligation Of The Four Bamhi Hindiii Restriction Fragments Of Pamp And Pkan Produc (1)

Ligation of the four BamHI/HindIII restriction fragments of pAMP and pKAN produces many types of hybrid molecules including plasmids composed of more than two fragments. However, only constructs possessing an origin of replication will be maintained and expressed. Three different replicating plasmids, with selectable antibiotic resistance, are created by ligating combinations of two BamHI/HindIII fragments:

• Ligation of the 784-bp fragment to the 3,755-bp fragment regenerates pAMP.

• Ligation of the 1,861-bp fragment to the 2,332-bp fragment regenerates pKAN.

• Ligation of the 1,861-bp fragment to the 3,755-bp fragment produces the “simple recombinant” plasmid pAK in which the kanamycin-resistance gene has been fused into the pAMP backbone.

1. Make a scale drawing of the simple recombinant molecule pAK described above. Include fragment sizes, the locations of BamHI and HindIII restriction sites, the location of origins(s), and the location of antibiotic-resistance gene(s).

2. Make scale drawings of other theoretical two-fragment recombinant plasmids with the following properties.

a. three kinds of plasmids having two origins

b. three kinds of plasmids having no origin

3. Ligation of the 784-bp fragment, 3,755-bp fragment, 1,861-bp fragment and 2,332-bp fragment produces a “super plasmid” pAMP/ KAN. Make a scale drawing of the super plasmid pAMP/KAN.

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