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Mini-Prep Plasmid Isolation and Identification
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Page 3-53 in lab manual & handout
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This is the second experiment (of a total of 3 experiments) for your molecular lab report.
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This experiment has two components
Mini-Prep isolate of plasmid DNA
Identification of plasmid DNA by gel electrophoresis (next week)
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Take cell and gently break them apart
Precipitate cellular debris in pellet
Save nucleic acids in supernatant
Precipitate nucleic acids to a pellet
Remove RNA
Gel Electrophoresis to confirm isolation
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Each group pick up the following tubes
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8
E. coli Lysis solution Potassium acetate Isopropanol RNAse Loading dye Cell Resuspension Buffer Plasmid suspension1xTris Buffer
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2. Isolation of Plasmid DNA:
Add 350 ul of lysis solution to cells
Gently mix, put on ice for 5 minutes
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1.
Centrifuge cells
Discard supernatant
Resuspend pellet in 200 ul resuspension buffer (tube-7)
“odd group numbers” will also add 5 ul RNAse
Incubate room temp. for 5 minutes
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2. Isolation of Plasmid DNA
Add 200 ul of potassium acetate to cell-lysis mixture
Mix gently
Place on ice 5 minutes
Centrifuge (14,000) for 5 minutes
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3. Isolation of Plasmid DNA
Carefully remove 0.5 ml of supernatant, place in new tube.
This contains your plasmid
Add 1.0 ml ice cold isopropanol
Place on ice for 10 minutes
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4. Isolation of Plasmid DNA
Centrifuge for 10 minutes
You should now see a pellet of nucleic acids (plasmid DNA + RNA)!
Carefully remove & discard all supernatant
Air Dry inverted for 10 minutes
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5. Isolation of Plasmid DNA
Add 60 ul of T.E. buffer to dissolve pellet
Remove 40 ul of this dissolved solution and add 5 ul of loading dye
To the remaining 20 ul of sample add 20 ul of T.E. Buffer and 5 ul of loading dye.
Store in freezer for next week.
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Next week
Confirm your isolation with gel electrophoresis
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