Rapid dna ligation kit

The positive PCR amplicons were ligated into a pGEM-T easy vector (Promega, Madison, WI, USA) using a Rapid DNA ligation kit (Promega), according to the manufacturer’s instructions. The recombinant DNA was transformed into the Escherichia coli DH5α competent cell and screened using the blue-white colony screening system. The positive colonies with an inserted COI gene were cultured on Luria–Bertani agar. The plasmid DNA containing the relevant DNA was extracted using an Invisorb Spin Plasmid Mini Kit (STRASTEC Molecular GmbH, Berlin, Germany), following the manufacturer’s instructions. The extracted plasmid was sequenced by the commercial service of Macrogen Inc., South Korea, using a universal forward T7 primer.

Calf intestinal alkaline phosphatase (CIP), T3 RNA polymerase, “High Fidelity” (PvuII and EcoRI) and other restriction enzymes, T4 polynt kinase (PNK), and MuLV RT were from New England Biolabs. DNase (deoxyribonuclease)-free RNase (ribonuclease), ribonucleotides, and deoxyribonucleotides were obtained from Roche. RNase free-DNase I was from United States Biochemical. Rapid DNA ligation kit, RNasin (RNase inhibitor), and the phiX174 HinfI digest DNA ladder was from Promega. Radiolabeled compounds were from PerkinElmer. Pfu DNA polymerase was from Stratagene. DNA oligonucleotides were from Integrated DNA Technologies. G-25 spin columns were from Harvard Apparatus. RNeasy RNA purification and the Plasmid DNA Miniprep kits were from Qiagen. X-gal was from Denville Scientific, Inc. IPTG and media were from Gibco, Life Technologies. All other chemicals were obtained from Fisher Scientific, VWR, or Sigma. HIV RT (from HXB2 strain) was prepared as described [64 (link)]. The HIV RT clone was a generous gift from Dr. Michael Parniak (University of Pittsburgh). This enzyme is a non-tagged heterodimer consisting of equal proportions of p66 and p51 subunits. Aliquots of HIV RT were stored frozen at −80°C and fresh aliquots were used for each experiment.