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

5. Ligation Reaction Mess
T4 Ligase
ATP

6. Bacterial Transformation
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

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

12. 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

13. 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°

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