T4 dna ligase

To construct a new type of recombinant hyaluronidase using human SPAM1 (hSPAM1) and bovine SPAM1 (bSPAM1) gene information, the plasmid pCXN2-bSPAM1 and -hSPAM1 gene fragments were used as a template (Fig. 3A). After performing the 1 st PCR using the above method, the two fragments were ligated with T4 DNA ligase in a total volume of 10 μl (Elpis Biotech, Korea). The 2 nd PCR was subsequently performed with a mixture containing 0.1 ul of the resulting ligation product, 0.5 μM of each primer (Table 2), and Pfu polymerase in a total volume of 50 ul. After a denaturation step at 94°C for 3 min, 35 cycles were carried out at 94°C for 1 min, 60°C for 30 sec, and 72°C for 90 sec. The target DNA fragment was obtained by gel purification of each PCR product inserted into the pCXN2 and sequenced.

Hep3B cells (human hepatocarcinoma cells) were maintained in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U ml -1 penicillin G sodium and 100 μg ml -1 streptomycin sulfate (Gibco), and cultured at 37 °C in humidified 95% air and 5% CO 2 . A 1239-bp fragment of the human MBL2 gene was prepared by PCR amplification using human genomic DNA as a template (forward primer: 5′-GCTAGGCTGCTGAGGTTTCTT-3′ and reverse primer: 5′-GGGCTGGCAAGACAACTATTAG-3′). The PCR products, haplotype (ht) 1 [G -554 A -431 G -225 ] and ht2 [C -554 A -431 G -225 ], were used as templates for the cloning construct. Each PCR product was gel-purified with an agarose gel purification kit (GeneAll Biotech, Seoul, Korea) and ligated into a TOPO vector (Invitrogen, Carlsbad, CA, USA). The plasmid was digested with KpnІ and XhoІ (Takara, Shuzo, Japan) and ligated into a pGL3-basic luciferase reporter vector (Promega, Madison, WI, USA) using T4 DNA ligase (Elpis Biotech, Daejeon, Korea). All constructs were confirmed by restriction enzyme analysis and DNA sequencing. Plasmid DNAs were prepared from these constructs using an Endo Free Plasmid Maxi Kit (Qiagen, Hilden, Germany), and concentrations and purity were assessed by UV spectrophotometry and agarose gel electrophoresis.