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Exam%203%20review - Overview of the Central Dogma DNA...

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Overview of the Central Dogma DNA RNA protein 5’ UTR 3’ UTR RBS (ribosome binding site) ATG stop codon Met (first amino acid) DNA replication (DNA polymerase) Transcription (RNA polymerase) Translation (ribosome)
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1. Receive two FP genes (in E. coli DH5- α cultures) 2. Purify plasmid DNA from bacteria 3. Quantify amount of plasmid (make 1 μ g / μ l stock) 4. Perform restriction digest 5. Transform plasmid DNA into E. coli JM109-DE3 6. Purify fluorescent protein from bacteria 7. Analyze your FPs (spectrophotometry, SDS-PAGE) pRSET-B + FP Fluorescent Protein Lab Experiments
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1. Receive two FP genes (in E. coli DH5- α cultures) 2. Purify plasmid DNA from bacteria 3. Quantify amount of plasmid (make 1 μ g / μ l stock) 4. Perform restriction digest 5. Transform plasmid DNA into E. coli JM109-DE3 6. Purify fluorescent protein from bacteria 7. Analyze your FPs (spectrophotometry, SDS-PAGE) pRSET-B + FP Fluorescent Protein Lab Experiments
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The FP’s are Recombinant Proteins Recombinant Protein: introduce a gene into a plasmid vector and express it • the gene is expressed in a host (bacteria, etc.) that would not normally express it Vector: a vehicle for propagating and maintaining a cloned fragment of DNA ( Clone = DNA isolated from the original organism) Plasmid: a type of vector • naturally occurring • small, circular, double stranded DNA • 2 - 15 kb • independent of bacterial chromosome ** Clone in E. coli DH5- α because it doesn’t express the protein - sometimes recombinant proteins can cause problems to the host ** Express in E. coli JM109-DE3 (more about this later…) (supercoiled plasmid)
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Elements of a Plasmid Vector (NOTE: 4. and 5. are specific to our vector, pRSET B) 1. Origin of Replication (pUC) where DNA polymerase binds and initiates replication 2. Selectable Marker (amp) Amp encodes a beta lactamase gene that breaks down ampicillin that would otherwise kill the cell 3. Multiple Cloning Site (MCS) Bunch of restriction sites that you can use to get your DNA clone into the vector 4. F1 ori Allows you to generate a ss copy of your insert for sequencing 5. T7 promoter Allows expression of your gene as a protein (only in cells containing T7 RNA polymerase )
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1. Receive two FP genes (in E. coli DH5- α cultures) 2. Purify plasmid DNA from bacteria 3. Quantify amount of plasmid (make 1 μ g / μ l stock) 4. Perform restriction digest 5. Transform plasmid DNA into E. coli JM109-DE3 6. Purify fluorescent protein from bacteria 7. Analyze your FPs (spectrophotometry, SDS-PAGE) Fluorescent Protein Lab Experiments pRSET-B + FP
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Plasmid Purification - How does it work? (this is the Alkaline Lysis method) Single-stranded bacterial DNA Cell debris Solution I : Glucose - makes hypertonic environment EDTA - chelates divalent cations (takes ions away from DNA-degrading enzymes) Rnase A - enzyme that degrades RNA Solution II : NaOH - very basic (high pH) - breaks open cell wall - denatures DNA
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Exam%203%20review - Overview of the Central Dogma DNA...

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