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JY BB JK JK

JY BB JK JK. CS LE MG AR. NG JS SJ YW MK. DS LL DH JB. DNA Cloning. DNA Frag. Cloning Vector. Cloning Vectors - Plasmid. Other Vectors Bacteriophage Cosmid YAC. Ligation T4 Ligase. CTGGCCA CATGGACCGGT. GTCAGGTAC CAGTC. T4 Ligase (ATP). GTCAGGTAC CAGTC.

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JY BB JK JK

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  1. JY BB JK JK CS LE MG AR NG JS SJ YW MK DS LL DH JB

  2. DNA Cloning DNA Frag Cloning Vector

  3. Cloning Vectors - Plasmid Other Vectors Bacteriophage Cosmid YAC

  4. LigationT4 Ligase CTGGCCA CATGGACCGGT GTCAGGTAC CAGTC T4 Ligase (ATP) GTCAGGTAC CAGTC CTGGCCA CATGGACCGGT Sticky End Ligation Blunt End Ligation

  5. Ligation Reaction Mess T4 Ligase ATP

  6. Bacterial Transformation Dead Colony Dead Dead Dead Colony Dead Colony Selection LB + Ampicillin Compentent E. coli

  7. Screening Transformants Step 1 Step 2 Insertional Inactivation LacZ gene Blue/White ccb gene Dead/Alive Size of Insert PCR + Electrophoresis Miniprep + RE + Electro

  8. PCR Cloning • Taq Polymerase ends • Adds extra Adenosine onto 3’ end • Generating a 3’ A overhang A PCR Product A TA cloning vectors + Ligase Topo cloning vectors

  9. Topo cloning vectors

  10. Experimental Outline PCR Reaction TOPO Reaction Bacterial Transformation Miniprep + RE digest Electrophoresis

  11. Topo Reaction • Mix • 4 μl PCR reaction • 1 μl Salt Solution • 1 μl Topo vector • Incubate 5 min at room temperature • Set up Transformation

  12. Transformation • Gently thaw TOP10 cells on ice – It is essential to keep cells on ice at all times. Even a few seconds at room temperature can ruin experiment. • Add 2 μl Topo reaction to cells and incubate on ice for 15 minutes. – mix gently by stirring with pipette tip, do not pipette up and down. • Heat shock cells for 30 seconds at 42° - immediately return to ice. • Add 250 μl room temp SOC media – cap tube and shake horizontally for 1 hour • Spread 10, 50, 100 μl of cells on prewarmed LB+amp plates. • Grow O/N at 37°

  13. Experimental Time Table • Wednesday 9/27 • Lecture on Restriction Enzymes • Thursday 9/28 • Topo Cloning Reaction • Transformation • Restriction analysis of Fragment • Solutions for DNA Isolation • Friday 9/29 • Check and refrigerate transformants • Monday 10/2 • Lecture on DNA Isolation • Wednesday 10/4 • Pick colonies – streak and start O/N cultures • Thursday • Miniprep Isolation of DNA • Restriction Digest • Gel Electronphoresis

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