Preparation of T-vectors

[from N. Hadjeb & G.A. Berkowitz (1996) BioTechniques 20, 20-22]
1. Incubate 10 ug of EcoRV-digested vector (e.g. pBluescript II KS) with 2mM dTTP and 5 U Taq DNA polymerase in 100 ul total volume of PCR buffer at 72oC for 2 hr.
2. Phenol/chloroform extraction 1X and ethanol precipitation. Resuspend the pellet in 10 ul TDW.
3. Ligate at 16oC for 16 hr using 2 U of T4 ligase.
4. Agarose gel purify the linear T-vector. Vectors without T-overhang will self ligate and migrate differently than the linear form.