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33 Cards in this Set
- Front
- Back
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WHAT DOES RECOMBINANT DNA TECHNOLOGY INCLUDE
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dna cloning, gene cloning, and molecular cloning
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what do restriction enzymes do
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cut the dna at a sequence specific sites (recognition sequence)
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what are the RE in bacteria
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endonucleases these are part of the bacterial host defense
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what are some of the antibiotic resistance genes plasmids have
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penicillin, ampicillin, kanamycin
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how is the recombinant DNA closed
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ligase
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what does LacZ do
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allows effective and controlled expression
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in the production of human insulin what are the A and B genes fused to
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lacZ
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in the production of human insulin the A and B peptides synthesized as products are fused to what?
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Beta-galactosidase
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in the production of human insulin what does beta-galactosidase do
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prevent product breakdown of the A and B peptides
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in the production of human insulin what does the Cyanogen bromide (cNBr) do
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cleaves the peptide products at the Met resulting in A and B fragments being isolated
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what is used to isolate A and B fragments in the production of human insulin
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cNBr
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in the production of human insulin how is the human insulin finallaly formed
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by mixing A and B allowing S bonds to form which holds the two chains
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what have scientists used premutated plasmids and restriction endonuecleases for
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creation of new cloning sites within plasmids
insertion of any gene into the plasmid for cloning (using the speceficity of restriction endonucleases) selection for genes using specific antibiotic resistance genes |
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what do Hind III and KpN I do
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make sticky ends
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what is Hind III
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hemophilus influenza
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what is Klebsiella pneumonia
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Kpn I
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what makes blunt ends
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Sma I
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how do bacteria take up plasmids
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by shocking or salts
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how are proteins produced in mammalian cells from plasmids
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plasmids must be transcribed in the nucleus
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what do mammalian cells require for the cloned gene to be expressed
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specialized DNA sequences in the plasmid
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how do mamallian cells take up plasmids
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electrical shock, salts, lipids, dextrans
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what are the twpo ways large scale synthesis of plasmid DNA can occur
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conjugation (self replication of plasmid)
replication of both the plasmid and bacteria |
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what criteria must a gene meet in order to be cloned, grown in bacteria or mammalian cells
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size of gene or its cDNA
modification to protein by cell delivery to the target tissue |
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what is large scale preparation of DNA
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large amounts of DNA for transfection, transformation, mutation
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what is miniprep for DNA
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small amounts of DNA for sequencing, transformation, restriction digestion
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in both large scale and miniprep for DNA what occurs
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transformation of plasmid into competent bacteria
isolation of colony in antibiotic containing agar plates growing up small cultures of the bacteria containing the palsmid lysis, column purification |
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what are the Quantitiy and quality checks of DNA
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spectrophotometric analysis
restriction enzyme digestion agarose gel electrophoresis |
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what happens in agarose gell electrophoresis
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DNA is loaded into the gell and the molecules migrate to the positive pole
DNA FRAGMENTS SEPERATE SHORTESTS MOVES FASTEST |
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what does spectrophotometric analysis do
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determine absorbance
determine concentration dilute DNA |
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what are the steps of subcloning
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preparation of vector
preparation of insert gel extraction ligation transformation into bacteria plate on antibiotic containing agar plates isolate colonies make mini preps verify by sequencing or restriction digestion |
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what type of DNA analysis is used in subcloning
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miniprep
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what are the steps in mutagenesis
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cloning the DNA of interest into a plasmid vector
plasmid DNA is denatured to produce single strands synthetic oligonucleotide w/ desired mutation is annealed to the target region extending the mutant oligonucleotide using a plasmid DNA strand as the template the heteroduplex is propagated by transformation in E coli |
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what occurs in polymerase chain reaction
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DNA is denatured
primers annealed to single strands DNA P reads and copies DNA repeat steps |
