T4 polynucleotide ligase

Basic DNA manipulations and molecular techniques were done using established procedures [19 ]. Extraction of DNA from agarose gels was done with a GeneJET extraction kit (Fermentas); plasmids were isolated with high pure plasmid isolation kit (Roche). All oligonucleotides used were synthesized at the Unidad de Síntesis of the Instituto de Biotecnología, Universidad Nacional Autónoma de México; all PCR amplifications were carried out using High Fidelity Taq polymerase (Invitrogen). Amplification protocols consisted of 30 cycles of 1 min at 94 °C, 1 min at variable temperature (depending on the primer combination), and 1 to 3 min at 68 °C. After amplification, PCR products were extracted with phenol and precipitated with ethanol. The DNAs were resuspended in Tris-EDTA buffer and digested with the appropriate restriction enzyme(s) to generate the required ends in the fragments. The DNA fragments were purified before cloning by isolating them from the agarose gel. For ligations, T4 polynucleotide ligase (Fermentas) was used. Plasmid transformation of E. coli was done using CaCl₂-competent cells. All plasmid constructions were verified by restriction analysis and PCR and, in most of the cases, by DNA sequencing.

The oligonucleotides used in this study were purchased from Unidad de Síntesis Química, IBT-UNAM. PCR amplification was carried out with recombinant TaqDNA polymerase (Invitrogen) and PFU (Fermentas) as specified by the manufacturer. PCR products were purified with the High Pure PCR Purification Kit (Roche). Vectors were purified with the High Pure Plasmid Isolation Kit (Roche). T4 polynucleotide ligase was used as indicated by the manufacturer (Fermentas).