Week 02 - Exp 4

Week 02 - Exp 4 - BIOL 519 Week #2 BACKGROUND: Experiment 2...

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Week #2 1 BACKGROUND: Experiment 2 & 3 - Replica Plating An additional method for rapidly testing large numbers of colonies on a variety of plates is replica printing. The starting material for replica printing is usually either a plate with lots of well isolated colonies, or a single plate onto which you have already patched (we will use the latter in this experiment). The idea behind replica printing is to press a material such as sterilized velveteen onto the surface of a plate with well-grown and isolated colonies or patches, and thereby pick up cells from each of the colonies/patches. The same piece of velveteen is then pressed onto the surface of one or more sterile plates, thereby transferring some of the cells onto the surface of each of the plates. The position of the colonies/patches will be the same on each of the plates. If you keep track of the “top” of each plate by marking with a small line, then you can identify the position where each colony/patch should be located on each plate, and determine whether each of the colonies/patches grew on each of the plates, or the color of the colonies on each plate. The advantage of replica printing over patching is speed - hundreds of colonies can be transferred to multiple plates in a matter of minutes. The disadvantage is that the replica printing process can somewhat smear the colonies on the plate - making it harder to identify the individual colonies - especially if the starting plate is a plate full of isolated colonies. Also, replica printing is best used with an appropriate number of colonies on a plate. For example, several plates with only 10 colonies each would probably be better patched onto 1 or 2 plates rather than replica printed onto 10 plates. On the other hand, an over crowded plate might become too smeared upon replica printing, and might also be better patched. In this experiment, you will be introduced to the technique by replica printing from the plate you patched last time onto a fresh LB plate. Experiment 4 - Select for Spontaneous Mutations The first step in many genetic analyses is to isolate mutations that affect a particular phenotype. Once mutations have been isolated, one can then compare the wild-type and mutant strains to learn about the function of the gene product. Obviously, the isolation and construction of mutations is of central importance in genetics. The first principle that this experiment will illustrate is the isolation of spontaneous mutations from a bacterial population. Spontaneous mutations are defined as those that are present in the population without the experimenter exposing the culture to a mutagenic agent. The advantage of isolating spontaneous mutations is that every different type of mutation will be represented in the population (as opposed to a mutagenized culture which may mostly contain a particular type of mutation such as mostly transitions - in which a purine is replaced by a purine, or a pyrimidine is replaced by a pyrimidine). The disadvantage
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Week 02 - Exp 4 - BIOL 519 Week #2 BACKGROUND: Experiment 2...

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