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.
1. Briefly centrifuge the T-vector and insert DNA tubes to collect contents at the bottom of the tube.
2. Set up ligation reaction as described above
* Molar ratio of PCR product: vector may optimization as described below
3. Mix the reactions by pipetting. Incubate the reaction 1 hour at room temperature.
Alternatively, if the maximum number of transformants is required, incubate the reactions overnight at 40°C.
1. Use GeneCraftLs T4 DNA Ligase in performing T-vector ligations. Other commercial preparations of T4 DNA Ligase may contain exonuclease activities that may remove the terminal deoxythymidines from the vector.
2. 5x Ligation Buffer (without ATP) contains: 250 mM Tris-HCl (pH 7.8); 50 mm MgCl2; 50 mM dithiothreitol.
3. It is very important to vortex the 5x Ligation Buffer before each use.
4. Longer incubation times will increase the number of transformants. Generally, incubation overnight at 4‹C will produce the maximum number of transformants.
5. Use 0.5ml tubes known to have ow DNA-binding capacity.
OPTIMIZING INSERT:VECTOR MOLAR RATIOS
The vector have been optimized using a 1:1 molar ratio of the Control Insert DNA to the vector. However, ratios of 8:1 and 1:8 have been successfully used. If initial experiments with your PCR product are suboptiomal, ratio optimizationmay be necessary. Ratios from 3:1 and 1:3 provide good initial parameters. The concentration of PCR product should be estimated by comparison to DNA mass standards on a gel. The T-vector is approximately 3kb and is supplied at 50ng/µl.
T-VECTOR MULTIPLE CLONING SITE REGION
pBluescript II SK (+)
1. Trower, M.K. and Elgar G.S. PCR cloning using T-vectors. Methods in Molecular Biology, Vol. 31: Protocols for Gene Analysis, 1994 Human Press Inc., Totowa, NJ
2. Mead, D.A. etal. (1991) A universal method for the direct cloning of PCR amplified nucleic acid. Biotechniques 9: 657-663
3. Marchuk, D.A. etal. (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19: 1154
4. Holton, T.A. and Graham, M.W. (1991) A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res.19:1156
5. Clark, J.M. (1988) Novel non-templated nucleotide reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Rec. 18:L 9677-9686