Ezna tm blood dna midi kit

Peripheral blood was collected from the enrolled subjects in tubes coated with EDTA. Genomic DNA was extracted from blood leukocytes using the EZNA TM Blood DNA Midi Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer protocol. All samples were stored at -20°C until use. SSHunter software was used for sequencing the X-chromosome, and Primer 5.0 software (Premier Biosoft, Palo Alto, CA, USA) was used to design primers.Briefly, the 12-µL polymerase chain reaction (PCR) included the following reagents: 50-200 ng genomic DNA, 2X PCR buffer, 15 mM MgCl 2 , 5 µM primers, and 0.5 U Taq polymerase (Tiangen Biotech, Beijing, China). The primer sequences and PCR amplification cycle parameters for the 5lociare listed in Table 1. For allele typing, 6% polyacrylamide gel electrophoresis and silver staining were used, and the alleles were named according to the number of repeats in the sequences based on the recommendations of the International Society of Forensic Genetics.

Peripheral blood (3-5 mL) was obtained from all patients in tubes coated with EDTA. Genomic DNA was extracted from blood leukocytes using an EZNA TM Blood DNA Midi kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer protocols. Primers for PCR and single-base extension were designed using the Assay Designer software package (Sequenom Inc., San Diego, CA, USA) (Table 1). SNP genotyping was performed using the SNaPshot SNP technology according to the manufacturer protocols. Primary data were analyzed by GeneMapper 4.1 (Applied Biosystems, Foster City, CA, USA). The genotypes were determined based on the nucleotide present at the SNP site, as visualized by one or two differently colored peaks in the chromatograms. For quality control, 5% of the recruited subjects were randomly genotyped twice by researchers in a blind manner, with a reproducibility of 100%.