DESCRIPTION
Cloning of PCR products using plasmid T-vectors is an easy and well established method(1-4). This method takes advantages of the terminal transferase activity of Taq polymerase (5). This enzyme adds a single deoxyadenosine base to the 3' end of their reaction products. These PCR products can be ligated directly into a vector containing compatible single T-nucleotide overhangs. The BS-SK-T-vector was produced by a very efficient procedure usingterminal deoxynucleotidyl transferase and ddTTP (4). The T-vector is prepared from 2961-bp vector pBluescript II SK(+) by cutting with ECO R I, filling recessed 3'terminal thymidine to both ends. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmids by preventing recircularization of the vector and providing a compatible overhang fpr PCR products generated by certain thermostable polymerases. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3'-ends of the amplified fragments.
The T-vector contain T7 and T3 RNA polymerase promoters flanking a multiple clonong region within the á-peptide coding region of the enzyme â-galactosidase. Insertional inactivation of the á-peptide allows recombinant clones to be directly identified by color screening on indicator plates. To increase the efficiency of the vector the tailed product was religated and the linear form was purified from an agarose gel. This vector cannot be used for cloning of PCR products generated with some DNA polymerases like Pfu DNA polymerase that does not exhibit any terminal transferase activity (for cloning of such PCR products use our pBS-ECO RV vector). However a simple incubation of such PCR products with Taq polymerase will add 3' nucleotide overhang, enabling the cloning of these products into T-vector.

CONCENTRATION
50 ng/µl

STORAGE TEMPERATURE
-20°C

REFERENCES
1. Trower, M.K. and Elgar G.S, 1994 Human Press Inc., Totowa, NJ
2. Mead, D.A. etal. (1991) Biotechniques 9: 657-663
3. Marchuk, D.A. etal. (1991) Nucleic Acids Res. 19: 1154
4. Holton, T.A. and Graham, M.W. (1991) Nucleic Acids Res.19:1156
5. Clark, J.M. (1988) Nucleic Acids Rec. 18:L 9677-9686