3730xl dna sequencer

Genomic DNA was obtained from peripheral blood leukocytes and purified by erythrocyte lysis with ACK buffer (0.15 M NH₄Cl, 10 mM K₂HPO₄, 0.1 mM EDTA). Leukocyte extracts were treated with proteinase K and subsequent extraction with basic phenol/chloroform/isoamyl alcohol (25:24:1). DNA was quantified by spectrophotometric analysis (Synergy 2, Biotek Instruments, Inc., Winooski, VT, USA) and used as template for PCR amplification and sequencing. A 569 bp region of IL1RL1 distal promoter was amplified using primers F8 (5′-CCTGTCAGCTTCTGAGAATTGCGTG-3′) and R8 (5′-CCAGTTTATCAGTTAAGAGACAGGAA-3′). PCR products were visualized on 1% agarose gel, then purified using QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) and sequenced with internal primers F9 (5′-GAAAGAAACACCAAATAAAGCAAC-3′) and R9 (5′-CCACATTCTCCCTATTTGAAAGATC-3′) using 3730XL DNA Sequencer (Macrogen Inc., Korea). Finally, SNPs were determined by analyzing the corresponding electropherograms with ChromasPro software v1.41 (Technelysium, Pty Ltd., Tewantin, QLD, Australia). Multiple alignments of the analyzed sequences were performed using Vector NTI Advance 10.0 software (IBI, New Haven, CT, USA).

A fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified with the primers F210-CO1 (5′-GTAATGCCAATTATGATTGG-3′) and COA (5′-AGTATAAGCGTCTGGGTAGTC-3′)87 . PCR reactions were performed in a total volume of 20 μl, using 8 μl of the REDEXtract-N-Amp Tissue PCR Kit (Sigma-Aldrich), 10 pmol of each primer, 8.4 μl of ultrapure water, and 1 μl of DNA extraction. Thermal cycling was performed in a dual thermal cycler as explained before at 96 °C for 90 sec; followed by 35 cycles of 96 °C for 20 sec, 48 °C for 80 sec and 72 °C for 90 sec, and a final extension of 5 minutes. Amplifications were visualised in agarose gels, and purification and sequencing of the PCR products were performed in a 3730xl DNA Sequencer at Macrogen services (www.macrogen.com) with the same primers used for amplification of the fragment. Sequences were edited and trimmed with MEGA v. 5.2 software88 (link) and aligned with CLUSTALW. Sequences with singletons were re-amplified and sequenced again. The sequences obtained have been deposited in GenBank (accession numbers: KX792505-KX792527, www.genbank.com).