Quick blood dna purification kit

DNA from test and control groups was isolated from peripheral blood lymphocytes using the Quick Blood DNA Purification Kit (EURX, Gdańsk, Poland). The protocol attached by the manufacturer was followed. The quality and quantity of the isolated genomic DNA was determined using Nanodrop (Thermo Scientific, Waltham, MA, USA). All samples were standardized by dilution to a final concentration of 25 ng/μL. The samples were stored at −20 °C until further analysis.

The study was approved by the Wrocław Medical University Bioethics Committee, Consent 641/2014, December 14, 2014. Blood (n = 109) from apparently healthy individuals was obtained following an informed consent according to the Declaration of Helsinki. Blood samples were collected on EDTA (for DNA extraction, flow cytometry and glycosphingolipid extraction) or on heparin (for RNA extraction), and washed RBCs were stored in CellStab low-ionic strength preservative solution (DiaMed, Cressier, Switzerland). DNA was extracted using Quick Blood DNA Purification Kit (EURx, Gdansk, Poland). RNA was prepared from the buffy coat using Human Blood RNA Purification Kit (EURx, Gdansk, Poland). The P₁/P₂ phenotype was determined by standard haemagglutination method using the human monoclonal anti-P1 antibody (S1 Table).

Genomic DNA was isolated from livers of sacrificed pigeons using Quick Blood DNA Purification Kit (EURx, Gdansk, Poland) according to the protocol provided by the supplier. Primers and conditions used in the PCR reaction for amplification of the A4GALT gene are listed in Supplementary Table S3A,B. PCR was performed using an MJ Mini gradient PCR apparatus (Bio-Rad, Hercules, CA, USA) in 100 µL reaction mixes containing 200 ng of pigeon genomic DNA (template), 0.2 mM dNTPs, HF Taq buffer with MgCl₂ (20× dilution), 0.2 mM forward and reverse primers, and 1 unit of HF Taq polymerase (Thermo Fisher Scientific, USA). The PCR products were purified using Gel-Out (A&A Biotechnology, Gdansk, Poland). The PCR products were further amplified using primers with restriction sites: XhoI (F) and NotI (R) (Thermo Fisher Scientific, Waltham, MA, USA) and cloned into the pCAG vector (kindly provided by Prof. Peter W. Andrews, University of Sheffield, Sheffield, UK) [18 (link)]. To test for the presence of A4GALT paralogs, the PCR products were digested with PaeI and HincII (Thermo Fisher Scientific, Waltham, MA, USA); 500 ng of purified PCR products were digested in 37°C for 6 h and analyzed by electrophoresis in 1% agarose gel with MIDORI green dye (Nippon Genetics Europe, Duren, Germany).