1. Cloning DNA, Plasmids and Transformations By: Stephen Sullivan and Julie Ethier

2. Key Words and Definitions Recombinant DNA- fragments of DNA composed of sequences originating from at least 2 different sources. Restriction Endonucleases- enzymes that are able to cleave double- stranded DNA fragments at specific sequences aka restriction enzymes Vectors- vehicles by which DNA may be intoduced into the host cell Host cell- a cell that has taken up a foriegn plasmid or virus and whose cellular machinery is being used to express the foriegn DNA Competent Cell- a cell that readily takes up a foriegn DNA.

3. DNA Cloning Overview 1. Generation of DNA fragments using restriction endonucleases. 2. Construction of a recombinant DNA molecule 3. Introduction into the host cell 4. Selection

4. What is a plasmid? Plasmids are small, circular, double-stranded DNA molecules lacking a protein coat that naturally exists in the cytoplasm of many strains of bacteria. Bacteria cell Plasmid DNA Circular chromosomal DNA Plasmid DNA are several thousand base pairs each, range in size from 1,000 to 200, 000 base pairs 4 million base pairs

5. What is transformation? Transformation is the introduction of DNA, by plasmids or viruses into a bacteria cell.

6. How are Plasmids Connected with DNA Cloning? Here is a short video to better explain PLASMIDS in DNA cloning http://www.scivee.tv/node/16947

7. Transformation Now that you know how plasmids work and how they are involved with DNA cloning we can look at TRANSFORMATION.... http://www.molecularstation.com/science-videos/video/61/plasmid- cloning/

8. Inserting a DNA sample into a Plasmid Plasmids are first collected from a bacterial cell and DNA is extracted. Plasmids and foreign DNA are cut by restriction endonuclease producing sticky and complementary ends. These two intermediates ends recombine due to complementary base pairing and are linked together by DNA ligase. A new plasmid containing the foreign DNA is formed.

9. DNA cloning using plasmids and transformation The small size of plasmids allows for bacteria to ably take the plasmid into its cell. This introduction of DNA from an external source is transformation. Once the bacteria/host cell has taken in the foreign DNA it is transformed. If the host cell easily takes foreign DNA then it is described as a competent cell. If not it can be chemically induced.

10. How is cloning DNA, plasmids and transformation used in the real world? Synthesis of insulin DNA plasmids can be very helpful in real life to manipulate DNA into synthesizing insulin, using plasmids and E. coli bacteria. Which can be used for people suffering from diabetes.

11. Stanley Cohen and Herbert Boyer's Animation video http://www.dnalc.org/resources/animations/transformation1.html

12. References Nelson Biology 12 DNA cloning. biotechnology learning hub. Nov 28, 2010. Phillips, Theresa. Essentials for Cloning and Studying Genes. About.com. Nov 28, 2010. Uppangala, Nidhi. Biotechnology and Types of Cloning. Nov 28, 2010 Inserting a DNA Sample into a Plasmid. Access Excellence. Nov 28, 2010