BIOS 390 ERIC KYLE WEEK 3 & 4 : LAB

BIOS 390 ERIC KYLE WEEK 3 & 4 : LAB DNA cloning is the process of making multiple, identical copies of a certain piece of DNA. A gene or other DNA fragment of interest is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that are able to “cut and paste” DNA, and then a molecule of recombinant DNA is produced. Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains. In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as “factories” to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics. The basic steps of DNA cloning: 1. Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA). 2. Transform the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid. 3. Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it.