Experiment 3: Cloning a DNA Fragment into a Bacterially-Derived Plasmid Vector Foregin DNA = 720 Plasmid...

Experiment 3: Cloning a DNA Fragment into a Bacterially-Derived Plasmid Vector

Foregin DNA = 720 Plasmid DNA 2804

Post-Lab Questions

1. What is the expected size of the plasmid plus the cut foreign DNA?

2524 base pairs

2. What type of ends do the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning?

3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does this enzyme work?

4. How would you clone a gene into a plasmid if there were no common restriction sites between the two DNA sequences?

Expert Solution

1. The expected size of the plasmid plus the cut foreign DNA would be 3,524 bp.

2. BamHI recognizes the sequence 5'-GGATCC-3' and EcoRI recognizes the sequence 5'-GAATTC-3', and both these enzymes produce sticky ends which means that they leave single-stranded overhangs. sticky ends are very useful in molecular cloning. If our gene of interest and the plasmid is cut by the same enzyme (eg- EcoRI), they will have matching overhangs, then the two overhangs can easily stick together by means of base complementarity. sticky ends also ensure that the DNA fragment is inserted into the plasmid in the right orientation. since the sticky ends hold the two pieces of DNA close together, it also facilitates easier ligation.

3. The enzyme DNA ligase is required to permanently link the digested foreign and plasmid DNA together. DNA ligase works by forming a covalent bond between the phosphate group of one strand and the deoxyribose group of another. Its mechanism of action has been depicted in the following diagram-

-

4. There are several techniques for generating a recombinant DNA molecule when a common restriction site between the Gene of interest and the vector is absent. some of these are-

- the Topoisomerase cloning technique

- Cre/lox P System

- Lambda recombinase.

gladiator answered 2 years ago

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