Joyce: T-tailing pBSKS

Concept
A T-tailed plasmid can be used to rescue DNA that have A-overhangs (as those generated using PCR with Taq polymerase - Taq has terminal transferase activity that adds a single adenosine to the 3'end of its substrate). To make the T-tailed plasmid, cut it first with a blunt cutter, then incubate it with Taq polymerase in the presence of TTP. In the presence of TTP alone, the terminal transferase activity will add a single T to the 3' end of the cut plasmid. This can then be used in a ligation reaction with the PCR products.

Procedure
1. Obtain about 11 ug of pBSKS

2. Cut with a blunt cutting restriction enzyme (like EcoRV) in a 100 ul volume

3. Remove a 10 ul aliquot to use as a control

4. Precipitate the remaining 90 ul (~10 ug) using NaOAc and ethanol

5. T-tail the DNA using:

10 ug of pBSKS 10 ul of 10X Taq buffer (no MgCl2) 0.5 ul 100 mM dTTP 6 ul 25 mM MgCl2 1 ul 5 U/ul Taq Sterile dH2O to 100 ul

5. Incubate at 72°C for 2 hours

6. Extract with 1X phenol and 2X ether

7. Precipitate the DNA with NaOAc and ethanol

8. Resuspend in 150 ul of sterile dH2O

9. Perform 2 sets of transformation controls as follows:

pBSKS-digested + ligase --> many colonies pBSKS-digested - ligase --> very few (indicates complete digestion was successful by showing                                          low background of undigested pBSKS)

T-tailed + ligase      --> very few (shows amount of digested pBSKS that doesn't have a T-tail) T-tailed - ligase      --> close to 0 (shows amount of undigested pBSKS in sample)