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Published byBaldric Summers Modified over 9 years ago
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Plasmid DNA Restriction Enzymes “cut” Plasmid DNA Piece of DNA is Removed New Piece (gene) of DNA is “stitched” to Plasmid DNA New DNA (gene)
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Bacterial Proteins that Cut Both Strands of the DNA Moloecules
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Small Ring of DNA Found in a Bacteria Cell Plasmid DNA E. Coli cell
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Bacteria Breaks Down Pollutants into Harmless Products Bacteria Extracts Minerals from Ores Insert the Human Gene into Bacteria to Produce Insulin for Diabetics Produce Artificial Sweeteners
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To transform the DNA of E. Coli bacteria by inserting a gene that will make the bacteria resistant to the antibiotic, ampicillin
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Ampicillin:A chemical that kills bacteria pGREEN:A plasmid that contains genes that protects bacteria from ampicillin and makes the bacteria turn grenn Plasmid: A circular piece of DNA found in bacteria LB plate: An Agar plate to grow bacteria on LB/Amp plate: An Agar plate that contains ampicillin LB broth: Food for bacteria
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1. Use micro-pipets and innoculating sticks to mix calcium chloride solution with E. Coli bacteria. 2. Label 4 Agar plates. LB +pVIB LB – pVIB LB/Amp + pVIB LB / Amp - pVIB 3. Mix pVIB plasmid with appropiate bacteria / CaCl 2 solution. 4. “Heat shock” bacteria in hot water bath and ice so that it takes in plasmid. 5. Spread + pVIB bacteria on “+” Agar plates 6. Spread – pVIB bacteria on “-” Agar plates 7. Incubate at room temperature for 72 hours and record bacterial growth.
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1. Practicing sterilizing technique 2. New tip for micropipette 3. Preparing calcium chloride solution
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4. Sterilizing inoculating stick 5. Scraping E. Coli bacteria 6. Adding E. Coli to CaCl 2 solution
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7. Inserting pVIB (plasmid DNA) into E. Coli 8. Inoculating Agar plates with genetically transformed E.Coli 9. Spreading bacteria evenly on Agar plates
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bacteria with gene antibiotics applied bacteria without gene antibiotics applied bacteria without gene normal growing conditions bacteria with gene normal growing conditions
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bacteria with gene antibiotics applied bacteria without gene antibiotics applied bacteria with gene normal growing conditions bacteria without gene normal growing conditions + pVIB LB/ Amp - pVIB LB/ Amp + pVIB LB - pVIB LB
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What are the controls for this experiment? Why is it important to practice sterilizing technique? Did the students successfully insert the pVIB gene? Why would you sometimes take antibiotics?
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