These methods were used each time a new intermediate plasmid was constructed. This process usually took between four to seven days, or longer, depending on a variety of factors.
- Digest DNA with restriction enzymes that cut at sequence-specific sites.
- Gel purify DNA to retrieve cleaved section, or clean DNA to rid sample of impurities.
- Ligate insert into new plasmid or religate digested plasmid.
- Transform ligated plasmid into E. coli, spread onto antibiotic-containing medium, and let colonies grow overnight
- Pick colonies and allow bacteria to grow overnight in nutrient-rich medium
- Miniprep cells to isolate the plasmid from the bacteria
- Verify the plasmid by digesting with appropriate restriction enzymes and then running on an agarose gel
- If the correct intermediate plasmid is obtained, use it to design a new plasmid, and begin with step 1
These are very general descriptions and a sample of the protocols I used this summer. Other methods were used, such as PCR and dephosphorylation, but were not used as frequently as the common cloning methods listed above.