Gfx pcr dna and gel band purification kit

The PCR products of AiV 3C/3D region were purified using the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s instructions. The purified DNA fragments were sequenced and analyzed. Phylogenetic analysis of AiV was conducted according to the resulting sequences’ alignment in 3C/3D region using the maximum-likelihood method (Kimura 2-parameter model) with MEGA X software (https://www.megasoftware.net. accessed on 24 April 2021). There was a total of 518 positions in the final dataset. Codon positions included were 1st + 2nd + 3rd + noncoding. All positions with less than 60% site coverage were eliminated, i.e., fewer than 40% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). Because AiV is a globally distributed virus, 27 AiV isolates from different regions were chosen for the evolutionary analysis. The sequences of these reference strains shown in Figure 1 were obtained from GenBank. Except the two Taiwanese AiV strains, 24 genotype A and 1 genotype B isolates from different regions and year were used, which included early and later isolated AiV strains from Japan, Brazil, China, France, Sweden, Tunisia, and Vietnam.

The presence of intl1 and intl2 integrase genes was also detected by PCR using primers listed in Table 1. Moreover, E. coli isolates harboring the integrase gene were the amplification of variable regions using primers, and the PCR product was sequenced with an automatic sequencer (Cosmogenetech, Deajeon, Korea) after purification using the GFX PCR DNA and Gel Band Purification Kit (Amersham Bioscience, Freiburg, Germany). The DNA sequence data were compared with those in the GenBank nucleotide database using the BLAST program available at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).

Preparation of plasmid DNA was performed using the Miniprep kit (Promega). DNA digestion with restriction endonucleases and separation of fragments in agarose gel electrophoresis were performed as described by Sambrook et al. [27 ]. Polymerase chain reactions were carried out in a Gene Amp PCR System 9700 (Applied Biosystems). The amplification reaction mixtures (100 µl) contained Phusion High-Fidelity DNA polymerase buffer, 10 pmol of each primer, 50 ng of DNA template, and 10 units of High-Fidelity DNA polymerase (Fermentas). The cycling parameters were 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s and finally 7 min at 72 °C. PCR products were purified using the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Bioscience), following the manufacturer’s instructions.