Akademik Veri Yönetim Sistemi
Transactions of the Institute of Molecular Biology & Biotechnologies, cilt.7, sa.1, ss.132-135, 2023 (Hakemli Dergi)
The need for the use of molecular biology reagents has been increasing day by day. In addition to commercially provided molecular biology reagents, some and/or most reagents could be also produced in specific investigation laboratories manually. In this study, we aimed to extract Taq DNA polymerase enzyme by ethanol precipitation method within a short time and cost-effectively. For this purpose, first, we cloned the pOpenTaq plasmid with Taq (polA) gene into Escherichia coli JM107 strain. Confirmed transformants were subjected to Taq DNA polymerase extraction. Transformants were grown at 37°C for 16 hours in LBB with ampicillin (100 µg/mL). Then, cells were induced by 1 mM IPTG supplement and incubated overnight again. Lysozyme (10 mg/mL; 1:1000 V:V) and DNaseI (10 mg/mL; 1:20000 V:V) treatments were used in the extraction steps. The purification was carried out with a total use of 70% ethanol. Totally 50 µL of protein was obtained after a three-day protocol. In SDS-PAGE assays, approximately 94 kDa protein was checked and then used in PCR analysis. Taq DNA polymerase was used in the amplification of tef1-α and 28SrDNA in Fusarium graminearum genome. Both genes were amplified from the F. graminearum genome by using a common PCR protocol. The findings obtained from this study showed that the ethanol precipitation method could be easily adapted to Taq DNA polymerase extraction within a short time in specific laboratories which involve common equipment.