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

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Presentation on theme: "JY BB JK JKCS LE MG AR DS LL DH JB NG JS SJ YW MK."— Presentation transcript:

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

2 DNA Cloning DNA Frag Cloning Vector

3 Cloning Vectors - Plasmid Other Vectors Bacteriophage Cosmid YAC

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

5 Ligation Reaction Mess T4 Ligase ATP

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

7 Screening Transformants Insertional Inactivation LacZ gene Blue/White ccb gene Dead/Alive Step 1 Step 2 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 A PCR Product TA cloning vectors + Ligase Topo cloning vectors

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

12 Topo Reaction 1. Mix 4 μl PCR reaction 1 μl Salt Solution 1 μl Topo vector 2.Incubate 5 min at room temperature 3.Set up Transformation

13 Transformation 1.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. 2.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. 3.Heat shock cells for 30 seconds at 42° - immediately return to ice. 4.Add 250 μl room temp SOC media – cap tube and shake horizontally for 1 hour 5.Spread 10, 50, 100 μl of cells on prewarmed LB+amp plates. 6.Grow O/N at 37°

14 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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