BIO 326R 1st Edition Lecture 15 Outline of Last Lecture I. Phenotype vs Genotypea. MutantII. Plasmidsa. Namingb. Host rangec. Replicating plasmidd. Plasmid copy numberi. RNA I, RNA II, RopOutline of Current Lecture I. Plasmid compatibilitya. Incompatibility groupsb. Partitioning systemII. Cloning into a plasmida. PCRCurrent LecturePlasmid Compatibility- Will two plasmids co-exist in a cell?- plasmid compatibility= plasmids grouped by incompatibilityo incompatibility groups if same partitioning system (more on this later), not compatible if same origin, not compatible (like pMB1 and ColE1)These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- if two plasmids share an origin, copy number is about halfo probability that all plasmids will be in one cell when it divides p= 2(1/2)^2n- n= copy number of the plasmid- can only use if there is no partitioning system (will get to later) copy number of 1: p= 0.5 50% chance, not very goodo plasmids can improve probability with partitioning systems =molecular system that ensures each plasmid is place into mother and daughter cells at division- Similar to the chromosomes and how they’re segregatedCloning DNA into a plasmid- Characteristics of cloning a plasmido Origin that replicates in E. coli often has a high copy numbero Antibiotic resistance markero Multi-cloning site=> stretch of DNA that contains high number of restriction endonuclease recognition sites Endonuclease recognition sites= location you clone DNA into- PCR= Allows you to make copies of a desire piece of DNA by amplificationo DNA polymerase tag (functions at a high temperature, 72 degrees Celsius) 95 degrees Celsius- 30s denatures DNA, makes single stranded 55 degrees Celsius- 30s allows primers to anneal 72 degrees Celsius- 30s DNA polymerase extension temperatureo Steps Amplify the gene Digest with restriction endonuclease- =bacterial enzymes that can cleave specific DNA sequences- Ex: 5’-G|AATTC-3’ 5’-G + AATC-3’ 3’- C TTAA|G-5’ 3’-CTTAA + G-5’ DNA
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