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.

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