Dna blood mini isolation kit

Blood collection and processing for VDR polymorphisms: Blood samples were collected into 4 ml of ethylenediaminetetraacetic acid (EDTA)-coated Vacuette. White blood cells (WBCs) were separated using RBC lysis buffer. DNA was isolated from WBCs using Qiagen DNA blood mini-isolation kit according to manufacturer's instructions. Forty micrograms of DNA was used for each reaction of polymerase chain reaction (PCR). PCR was performed using PCR Master Mix (Fermentas, USA), and restriction fragment length polymorphism (RFLP) was performed using the respective restriction endonuclease along with buffer (Fermentas, USA). Fok1, Bsm1, Apa1, and Taq1 polymorphisms were performed based on the PCR-RFLP method as described by Park et al. [ 7 ] and Shabazi et al. [ 8 ] Poly A polymorphism was performed based on the PCR-Single-strand conformation polymorphism (SSCP) method described by Colagar et al., [ 9 ] with slight modifications.

The genomic DNA from lung cancer patients was extracted from 2 ml peripheral blood lymphocytes using Qiagen DNA blood mini-isolation kit (Germany) according to the manufacturer instructions. The blood sample was withdrawn before and after chemotherapeutic drug administration. Quantification of the genomic DNA samples was performed on a nano-drop spectrophotometer (Agilent Technologies, Santa Clara, USA). Total reaction volume of 25 μl (2X PCR master mixes, Sigma Aldrich, St. Louis, USA) and 50-100 ng of genomic DNA with 10 pmol of primers to set up the reaction with positive and negative controls in a thermal cycler 8800 (Agilent Technologies, Australia). The primer sequence, restriction enzymes, and genotyping method for seven genetic polymorphic genes included in the study are described in Table 1. The genotyping of XRCC1 codon 399 performed according to the earlier described method[ 26 ] XPD codon 751 genotyping performed according to the earlier described method.[ 27 ] For GSTs (GSTM1 and GSTT1), genotyping performed as a previously described method.
[ 28 ] Cytokine gene polymorphism of interleukin-1 (IL-1) β and IL-1RN performed by the earlier described method.[ 29 ] Five percent of the samples were randomly selected and genotyped, and we got 100% concordant results. T1-17 Table 1: Single nucleotide polymorphisms included in follow-up study and their location

Genomic DNA was extracted from 2 ml of the peripheral blood lymphocytes of lung cancer patients and controls using Qiagen DNA blood mini isolation kit (Germany) according to the manufacturers' protocol and its concentration quantified on a NanoDrop spectrophotometer (Agilent Technologies, Santa Clara, USA).