Cloning a Gene with His Tag & TEV Cleavage Site

How can I clone a gene into a plasmid vector with an N-terminal his tag and TEV cleavage site between the tag and the start of the sequence?

To clone a gene into a plasmid vector with an N-terminal His tag and TEV cleavage site, you would follow a series of steps involving molecular biology techniques. Here is a detailed guide on how to perform this cloning:

1. Obtain the necessary materials:
– Plasmid vector with an appropriate selection marker (e. g. , ampicillin resistance)
– Restriction enzymes appropriate for your gene and vector
– PCR primers specific to your gene of interest
– DNA polymerase for PCR
– DNA gel extraction kit
– DNA ligase
– Competent bacterial cells for transformation
– Appropriate culture media and reagents for bacterial growth and selection

2. Design primers:
– Design PCR primers that include your gene of interest and specific sequences to introduce the His tag and TEV cleavage site. – Incorporate restriction enzyme recognition sites at the ends of the primers, which match the sites in the plasmid vector that you plan to use

3. PCR amplification:
– Use the designed primers to amplify the gene of interest from a DNA template (e. g. , genomic DNA or a cDNA clone). – Perform PCR with high-fidelity DNA polymerase to reduce the likelihood of introducing errors. – Verify the PCR product by running an agarose gel electrophoresis

4. Plasmid preparation:
– Isolate the plasmid vector of interest from a bacterial culture using a plasmid extraction kit. – Verify the quality and quantity of the extracted plasmid by analyzing it on an agarose gel or using a spectrophotometer

5. Digestion of plasmid vector and PCR product:
– Incubate the extracted plasmid vector with the appropriate restriction enzymes that cleave the vector at desired sites. – Digest the PCR product similarly using the same restriction enzymes. – The restriction sites should be positioned such that the His tag and TEV cleavage site can be incorporated between the tag and the gene of interest

6. Purification of digested fragments:
– Purify the digested vector and PCR product separately using gel extraction or PCR purification kits. – This will remove any remaining enzymes and undesirable DNA fragments

7. Ligation:
– Combine purified digested vector and PCR product in a ligation reaction tube. – Add DNA ligase and buffer according to the manufacturer’s instructions. – Incubate the reaction mixture at an optimal temperature for DNA ligation

8. Transformation:
– Introduce the ligation mixture into competent bacterial cells (e. g. , E. coli) using a suitable transformation method (e. g. , chemical transformation or electroporation). – Plate the transformed cells on a selective agar plate containing the appropriate antibiotics for the selection marker on the plasmid vector

9. Screening and confirmation:
– Select colonies that grow on the selective media, indicating successful transformation. – Perform colony PCR or miniprep to confirm the presence of the desired insert in the plasmid. – Verify the correct sequence of the cloned gene and the presence of the His tag and TEV cleavage site by DNA sequencing

By following these steps, you should be able to clone your gene of interest into a plasmid vector with an N-terminal His tag and TEV cleavage site. This cloned construct can then be used for various downstream applications, such as protein expression and purification

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