Subcloning Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Subcloning protocol

The following is a brief protocol of the subcloning procedure:

1. Analyze the restriction sites of the vector from which you would like to move your gene to your target vector - the restriction sites should be present 5' to 3' in the same order for both vectors.
2. Make sure that your gene(s) does not contain any of the restriction sites that you choose to cut out your gene with.
3. Digest out the gene from the original vector with restriction enzymes according to the instructions given, and separate the gene from the vector by running on a gel, cutting out the desired band from viewing under a UV light, and performing a gel extraction. (eg Qiagen).
4. Digest out the restriction sites from the target vector, and purify out the vector to remove the DNA between the restriction sites.
5. Perform a ligation with the cut out gene and the target vector.
6. Transform the desired amount of ligated product into competent bacterial cells.
7. Select the desired colonies and grow up the bacteria in overnight cultures.
8. Purify the plasmid (eg can use commercial kits).
9. Store the purified plasmid at -80 degrees C or -20 degrees C in aliquots.
10. If a ligation where the same restriction enzyme has been used on either side of the insert has been performed, it is necessary to check with other restriction enzymes or by sequencing to ensure that the insert has been inserted in the proper orientation.