You want to clone the mouse β-globin gene into an expression vector. Outline the steps you would take to insert this gene into the plasmid and how you would identify the recombinant vector. Use diagrams to show the steps and name the enzymes you would use.
1. Isolation of the mouse β-globin gene: The mouse β-globin gene needs to be isolated from genomic DNA using PCR or other methods.
2. Selection of plasmid: Choose an appropriate expression vector with a promoter suitable for expression of the β-globin gene in the desired host organism. For this example, we will use the pUC vector.
3. Preparation of vector: The pUC vector needs to be prepared by digestion with a restriction endonuclease that leaves an overhang that is compatible with the overhang generated by the restriction endonuclease used to digest the mouse β-globin gene. EcoRI and HindIII are used to cut the plasmid and the mouse β-globin gene, respectively.
4. Ligation of the gene into the vector: The β-globin gene and pUC vector should be ligated using T4 DNA ligase. This results in a hybrid plasmid in which the β-globin gene is inserted into the multiple cloning site (MCS) of the expression vector.
5. Transformation: The newly constructed plasmid with the β-globin gene is now introduced into competent cells of the desired host organism.
6. Selection of transformed cells: The transformed cells are then placed in a selection media which consists of antibiotics (ampicillin) to identify cells that have taken up the plasmid.
7. Screening of recombinant cells: The recombinant cells must be identified by screening the transformed cells to confirm that the mouse β-globin gene is present in the new plasmid construct.
8. Verification of recombinant vector: The final confirmation of successful gene cloning is done by sequence analysis to verify that the mouse β-globin gene has indeed been cloned and inserted into the plasmid.
The diagram below illustrates the steps involved in cloning of the mouse β-globin gene into an expression vector:
<img src="https://i.imgur.com/FUsfQlI.png" alt="Diagram of Cloning Mouse β-globin gene into Expression Vector" width="600"/>