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Lecture

Restriction Enzymes are Enzymes

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Department
Biochemistry
Course
Biochemistry 2280A
Professor
Eric Ball
Semester
Fall

Description
Restriction Enzymes are EnzymesLike any other enzyme EACH restriction enzyme has preferred conditions in which it functions These include temperature pH salt concentration Restriction Enzymes have reaction kinetics Takes time for it to work Certain amount of DNA amount cleaved over time Linearconstant relationship over time positivewill plateau eventually when they are all cleaved DNA digested over timeWe can cut DNA with restriction enzymes How can we stitch DNA back togetherDNA Ligase glue for DNA will reseal compatible sticky ends and much less efficiently blunt ends requires an energy source ATPSteps In CloningDNA CloningPCR digest 100ng of YFG and a plasmid vector with the same restriction enzyme or on that gives compatible overhanging sequencesHow do we get the restriction enzyme cut sites on YFG 1 putting EcoRI sites on the cDNA by incorporating the sites into PCR primers2 inactivate the restriction enzyme 3 Incubate vector plasmid and insert YFG together in the presence of DNA ligase and ATP ligation step the sticky ends anneal hybridize ligase forms covalent bonds phosphodiester bonds to seal the ends 4 Need to get the plasmid into ecoli Introduce the ligated plasmind in EcolyTransformationL Natural property of some bacteria Ecoli have to be treated with chemicals to do it well mix Ecoli with ligated DNA some of the Ecoli will take up the plasmids be transformed5 Plate cells onto solid media agar containing antibiotic ampilicillin Single ells that have taken up the plasmid will grow forming visible colonies blueNot all of the transformed bacteria contain a plasmid that has YFG inserted What isare the reasons for this A Spontaneous ampicillin resistant colonies may appear B The plasmid vector can recircularize without YFG C Contaminating DNA may be ligated into the vector D All of the above E None of the above D All potentially correct Aunlikely but can happen
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