BMI_Lab8DNAPurification - Laboratory VIII Topic:...

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Laboratory VIII Topic: Purification of DNA, plasmid isolation using alkaline method Introduction : Genetic transformation occurs when bacteria take in plasmid DNA and express the genes encoded by the plasmid. This genetic change often provides the organism with a new trait or characteristic by which the transformed cell can be identified or selected. Last lab exercise, pUC19 plasmid was transformed into DH5 α cells. The bla gene present in pUC19 plasmid produces beta-lactamase enzyme. The beta-lactamase enzyme can destroy beta-lactam antibiotics such as ampicillin and penicillin. Note: Antibiotics are used to kill bacteria In non-selective media (LB media without ampicillin), both transformed and non-transformed DH5 α cells grow well and the numerous colonies formed coalescence into a bacterial lawn on the plate. Non-transformed DH5 α do not survive in selective media (LB media with ampicillin) while transformed DH5 α appear as translucent colonies of various sizes. Each colony represents a single transformed DH5 α cell (clone ). Transformation efficiency is a measure of the number of bacterial cells that are able to take up DNA molecules. Plasmids also have an origin of replication ( ori ) which allows plasmids to self replicate independently of the chromosomal DNA. Large quantities of pUC19 plasmid can be isolated by growing an overnight culture of a transformed colony in LB broth containing ampicillin. The pUC19 plasmid multiplies along with the bacterial cell. Any bacteria that do not have the plasmid die because ampicillin is added to the LB broth. Plasmids have a supercoiled circular structure and are very small in size compared to chromosomal DNA. Hence plasmid DNA is easily separable after alkaline lysis of cell pellet. Today we will isolate the pUC19 plasmid from the transformed DH5 α. We use several solutions for isolating the plasmid DNA. P1 buffer: contains 50mmM of Tris HCL and 20mM Sodium EDTA at pH 8. Tris is a buffering agent used to maintain a constant pH (= 8.0). EDTA binds divalent cations (Mg +2 and Ca +2 ) that are necessary for DNAse activity and for cell membrane intergrity. P2 solution:
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This note was uploaded on 04/24/2008 for the course PHS 2301 taught by Professor Carvalho during the Spring '08 term at St. Johns Duplicate.

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BMI_Lab8DNAPurification - Laboratory VIII Topic:...

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