Here is a brief protocol subcloning procedure:
- Analyze the vector restriction sites from which you want to move your genes for your target vector – the restriction site must be present 5 ‘to 3’ in the same order for both vectors.
- Make sure that your gene (s) do not contain any of the restriction sites that you choose to cut your genes.
- Digest out of genes from the original vector with restriction enzymes in accordance with the instructions given, and isolate the gene from a vector by walking on a gel, cut the desired band of viewed under UV light, and extraction of gel. (For example Qiagen).
- Digest exit restriction sites of the vector targets, and purify out the vector to remove the DNA between the restriction sites.
- Perform ligation by cutting out the gene and target vector.
- Transform the desired amount of product bound to the bacterial cells are competent.
- Select the desired colonies and grew the bacteria in a culture overnight.
- Purify plasmid (eg to use commercial kit).
- The purified plasmid Store at -80 ° C or -20 degrees C in aliquots.
Where ligation 10. If the same restriction enzymes have been used on both sides of the insert has been done, it is necessary to check with other restriction enzymes or with an order to ensure that the insert has been inserted in the proper orientation.