Adult C. Sinensis Worms For The Extraction Of Genomic Dnas Essay example

1368 Words Oct 6th, 2016 6 Pages
Adult C. sinensis worms for the extraction of genomic DNAs in this study were prepared as previously described (Bae et al. 2001). O. viverrini adults were kindly gifted by professor Woon-Mok Shon in the Department of Parasitology, Gyeongsang National University, Korea. Genomic DNAs were extracted from individual worms with the Wizard DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturers’ instructions. Live C. sinensis adults, collected from experimental rats and infected with C. sinensis metacercariae (Kim et al. 2015) were applied for the extraction of total RNAs using TriZol reagent (Invitrogen, Carlsbad, CA, USA) following the standard protocol. The guide to the care and use of experimental animals was approved by the Institutional Review Board and conducted in the Laboratory Animal Research Center of Gachon University (protocol GIACUC-R2014005).

Southern- and Dot-blot Hybridization Assays Genomic DNAs (each 5μg from 30 individuals) of adult C. sinensis and O. viverrini worms were digested with Hind II and fractionated through 0.8% agarose-gel electrophoresis. The restricted DNA fragments were transferred onto nylon membranes (Hybond-N+; Amersham Pharmacia Biotech, Uppsala, Sweden) by capillary action in 10 x standard saline citrate (SSC) buffer. The blots were hybridized with CsRn1 probes enzymatically labeled with an ECL Direct Labeling Kit (Amersham Pharmacia Biotech). The labeling and hybridizing conditions followed the manufacturer’s…

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