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

Ligation Reaction Mess T4 Ligase ATP

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

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

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

Topo cloning vectors

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

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

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°

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