1. Cloning Vector Map

2. Vector Characteristics
Topoisomerase
Contains a T-overhang
Linked topoisomerase
Topoisomerase

3. PCR Fragment Properties
PCR Product A
Contains an A-overhang
A-T base pairing helps anchor the insert to vector

4. Ligation Mix Single 3´ thymidine (T) overhangs for TA Cloning
Topoisomerase
PCR Product A
Single 3´ thymidine (T) overhangs for TA Cloning
Taq DNA polymerase adds nontemplate dependent A at the 3’ end
Topoisomerase covalently bound to the vector ("activated“ vector)

5. How it Works?

6. Recombinant Construct

7. Transformation
Competent Cells (E. coli
Heat Shock Method

8. Cloning