Accuprep dna extraction kit

Genomic DNA was extracted from 4 ml of peripheral blood mixed with ethylenediaminetetra acetic acid (EDTA) using an AccuPrep DNA extraction Kit (Bioneer, Daejeon, Republic of Korea): After 20 μl of proteinase K and 200 μl of lysis buffer (200 mM Tris–HCl, 25 mM EDTA, 300 mM NaCl, 1.2% sodium dodecyl sulfate) were added to peripheral blood leukocytes, the mixture was incubated at 60 °C for 10 min. The lysate was extracted with an equal volume of isopropanol. After washing with 500 μl of washing buffer and ethanol, the pellet was dried and suspended in 200 μl of sterile distilled water. DNA extracts were stored at − 20 °C.

The genomic DNA was extracted by using a commercial bacterial DNA extraction kit (AccuPrep^® DNA Extraction Kit (Bioneer, Daejeon, Korea) according to the kit manufacturer’s instructions. Briefly, 1 mL of STEC isolate (grown overnight in LB broth; Difco^TM Becton and Dickson and Company) was pelleted in Eppendorf tube by microcentrifuge at 10,000× g at max speed for 5 min. The supernatant was discarded followed by the addition of 200 µL of CL buffer and vortexed to completely resuspend the cell pellet. Then, 20 µL of proteinase K solution was added, samples were vortexed, and the cell solution was incubated at 56 °C for 15 min. After the lysis, 200 µL of BL buffer was added and vortexed and tubes were incubated at 70 °C for 10 min. The genomic DNA was concentrated by the addition of 200 °C for 15 min of absolute ethanol, pulse vortexed and centrifuged at 6000× g for 1 min. This was followed by the addition of 600 µL of BW buffer and 700 µL of TW buffer for washing and centrifuged at maximum speed for 1 min, respectively. The purified DNA was eluted in a fresh 1.5 mL microcentrifuge tube using 100 µL of elution buffer, kept at room temperature for 1 min, and centrifuged at full speed for 1 min. Finally, the DNA concentration was evaluated using a nanodrop spectrophotometer before being stored at −20 °C.