Tissue dna purification kit

The antibodies used in this study were as follows: mouse monoclonal anti-Cx45 (MAB3100, Merck Millipore, Billerica, MA, USA), mouse monoclonal anti-Cx43 (C13720, BD Transduction Laboratories™, San Joes, CA, USA), goat polyclonal anti-actin (C-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and goat anti-mouse IgG (H + L) HRP-conjugated (62-6520, Thermo Fisher Scientific, Rockford, IL, USA) antibodies. RNeasy^® Mini Kit (74104, Qiagen, Hilden, Germany), PrimeScript TM 1st strand cDNA kit (6110A, Takara Bio, Shiga, Japan), Taq polymerase PCR kit (DT95-E500, Solgent, Daejeon, Korea), tissue DNA Purification Kit (CME0112, Cosmogenetech, Daegu, Korea), T7 endonuclease 1 (T7E1) (M0302L, New England Biolabs, Ipswich, MA, USA), and LY (L0144, Sigma-Aldrich, St Louis, MO, USA) were purchased from local vendors.

For DNA extraction, ethanol was removed from fixed specimens (99% EtOH) by washing with distilled water, and DNA was extracted using a tissue DNA purification kit (COSMO GENETECH, Co. Ltd, Korea). DNA was extracted from individual specimens. mtCOI DNA was amplified in 20 μl reaction volumes containing extracted tissue DNA and primers LCO-1490 (5'-GGT CAA CAA ATC ATA AAG ATA AAG ATA TTG G-3') and HCO-2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') (Folmer et al. 1994 (link)). PCR conditions comprised initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 46 °C for 2 min, and extension at 72 °C for 3 min. This was followed by a final extension step at 72 °C for 10 min. PCR products were evaluated by electrophoresing amplification products on 1% agarose gel containing ethidium bromide. Purification of amplified products was performed using a PCR purification kit (COSMO GENETECH Co. Ltd, Korea), and both strands were sequenced using an ABI 3730XL sequencer (COSMO GENETECH Co. Ltd, Korea).

Before preparing the microscopic slides, genomic DNA was extracted from one leg of each specimen using a Tissue DNA Purification Kit (Cosmogenetech Inc., Seoul, Korea) according to the manufacturer’s instructions. The COI barcode fragment was amplified using two universal primers: bcdF05 (5′-TTTTCTACHAAYCATAAAGATATTGC-3′) and bcdR04 (5′- TATAAACYTCDGGATGNCCAAAAAA-3′) under the following conditions: 2 min at 94 °C; 40 cycles at 98 °C for 15 s, 50 °C for 30 s, and 68 °C for 60 s; and a final extension at 68 °C for 5 min (Dabert et al. 2008 (link)). The amplified products were sequenced using an ABI3100 automated sequencer (Perkin Elmer, Foster City, California, USA). Sequence assembly, alignment, and trimming were performed using Geneious 8.1.9 software (Kearse et al. 2012 (link)). We obtained a 654 bp fragment sequence of the COI gene from two individuals per mite species.