Lab 4 - Lab 4 Cloning Complementation Photosynthesis...

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Lab 4 Cloning Complementation Photosynthesis
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Cloning: Screening (ampicillin resistance & β -gal activity) Source DNA Plasmid DNA LacZ gene Encodes β - Galactosidase (blue product) Multi cloning site contains unique restriction sites Antibiotic resistance gene (amp R ) Origin of replication (induces replication in E. coli ) Plasmid (“shuttle molecule”) Transform & screen
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, Sequencing : Pick just one white colony
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Cloning: Remember: It’s important what kind of molecules you start with! If you use just one type of plasmid to transform bacteria you only get two different kind of bacteria: >99.99 % of bacteria without a plasmid (responsible for the lawn if there is no antibiotic in the LB plate) and <0.01% that took up the plasmid (responsible for isolated colonies if there is an antibiotic in the LB medium). If you use a “ligation reaction” (contains 1: relegated plasmid, 2: plasmid with insert X, 3: plasmid with insert Y, 4: plasmid with insert X+Y, 5: … all depends on the number of insert fragments generated by one or multiple restriction enzymes) for the transformation you get a mixed population of bacteria: >99.99 % of bacteria without a plasmid and <0.01% that took up the various plasmids. You then need to screen for the right clone (colony) using the appropriate selectable markers (antibiotics for the presence of an antibiotic resistance gene, X-Gal for an insert that disrupts the beta-Galactosidase gene = white colonies vs blue) You pick the right colony, amplify it in LB medium (with the correct antibiotic) and then isolate the plasmid (a lot of them; GMB1 exercise). You can then analyze the purified plasmid DNA to confirm that you work with the correct clone. You can digest the plasmid with restriction enzymes and separate these by gel electrophoresis. The sizes of the DNA bands in the gel give you information about the number of DNA base pairs in these fragments. You’ll see bands for the plasmid (relaxed, coiled and supercoiled) and band(s) for the DNA insert(s). The band pattern in the gel depends on the restriction sites present in the DNA and the restriction enzymes used. More in the GMB1+2 labs Further analysis includes DNA sequencing. More in the GMB1 lab.
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Complementation 1N yeast spores divide by mitosis 2N yeast cells divide by 1) mitosis 2N yeast cells divide by 2) meiosis Mating types: a with α e.g. grow on SD -His plates e.g. grow on YPD plates (incl. His)
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His biosynthesis in 1N spores b’ c’ d’ e’ f’ g’ h’ Intermediate products Enzymes Mutant gene = no functional enzyme = no growth Remember: DNA PreRNA mRNA protein (functional unit) Substrate accumulates Auxotrophs need His Prototrophs can make it Chemicals only rescue if they mimic intermediate products downstream of the mutation (high # of rescue chemicals = early in pathway
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This note was uploaded on 10/29/2009 for the course BIO biology taught by Professor Meighan during the Fall '09 term at University of California, Berkeley.

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Lab 4 - Lab 4 Cloning Complementation Photosynthesis...

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