![]() ![]() To achieve this goal we are going to do a "colony PCR". We need a way to find the colonies that are expressing our gene of interest off the vector plasmid. That can happen when, during ligation, the plasmid DNA annealed back on itself. There are a small proportion of bacteria on your selection plates that have a plasmid without the gene of interest. However, knowing that the bacteria growing in your broth or on your plate with ampicillin all have the vector plasmid responsible for amp resistance, that does not mean that these bacteria also have our gene of interest insert. This allows us to take bacteria from a colony and sub-culture them in liquid media to make millions of identical copies. Because bacteria reproduce asexually and are immobile on solid media, if you have an isolated colony on a plate, it is likely that the hundreds of thousands of bacteria making up that colony are daughters of a single cell (genetically identical). This process of using a marker (usually antibiotic resistance) to differentiate transformed cells from those not transformed is called selection. You must be careful to pick ONLY the bigger, central colony and not the satellites. This happens because the ampicillin in the media is destroyed in the area immediately around the colony secreting the enzyme therefore, there is no ampicillin in the area around the transformant and non-transformed cells can grow and divide enough to form smaller, satellite colonies. This enzyme is a secreted, soluble protein, which means that there may be smaller, non-transformed, "satellite" colonies around a true transformant. However, the amp resistance gene on the plasmid encodes an enzyme called beta-lactamase. Because only transformed bacteria are resistant to ampicillin, if we grow the bacteria on or in a medium containing ampicillin, those bacteria that did not take up plasmid DNA should not be able to reproduce to form colonies while those that express plasmid gene products and transfer the plasmid to their progeny will form colonies. In theory, any colony of bacteria growing on your LB+amp plate should contain a vector plasmid because the gene for antibiotic resistance is not chromosomal, but expressed from your plasmid. Lab 7: Series 3- Reverse Genetics: Picking Your Transformant 3 Outline of Experimental Design for REVERSE Genetics Project.2 To do on the day before the next lab:.1 Lab 7: Series 3- Reverse Genetics: Picking Your Transformant.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |