Question

Question 4

C. Cloning and restriction enzyme digest Video aid 1: Plasmid cloning Video aid 2: Restriction enzyme digest analysis The PCR was a success and your target region of 440 bp in length has been amplified. You igate a short linker containing an Apal restriction enzyme site onto both ends of the PCR product, digest it with Apal, and clone it into the Apal site of the 5 kb plasmid diagrammed below Bamll 300 EcoRI 3400 1000 2000 5 kb EcoRI Apal 1. List three components a cloning vector must contain A cloning site A replication origin A selectable marker-Drug resistant gene 2. Draw the end of the fragment that will be generated after digestion with the Apal enzyme. Indicate the type of ends generated by this enzyme (Blunt/Sticky, 5 or 3 overhang) (3) 3 ctccGG G5 It forms sticky ends with 3' overhang ends 3. What is the size of the plasmid after your PCR product has been successfully ligated into the 5440Bp 4. Draw the plasmid containing the cloned insert. Indicate clearly where the insert will be located. Include RE sites and distances 5.44kb 300 2000 Clone Apal Ecoll English (South Africa)

Answer 1

300 BamHI EcoRI 3400 1000 EcORI INSERT

The insert is cloned into the ApaI enzyme site.

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