Ep gradient pcr

Genomic DNA was purified using a DNA isolation kit (Sigma Aldrich, USA). Total RNA and plasmid purification was done using Trizol reagent (Invitrogen, USA) and GenElute plasmid extraction kit (Sigma Aldrich, USA) respectively, according to manufacturer's instructions. Concentration and purity criteria of the nucleic acids were quantified in a BioPhotometer Plus (Eppendorf, Germany). Primers used for amplification of direct PCR for algC and RT-PCR of internal regions of algC are listed in Table 1. Primers were designed using Primer Quest (IDT, USA) and synthesized (Sigma Aldrich, USA). For amplification of the 1781 bp fragment algC from genomic DNA, PCR conditions used were initial denaturation of 5 min at 94°C, followed by 30 cycles of 30 sec at 94°C, 20 seconds at 54.5°C, and 45 seconds at 72°C with final extension of 5 minutes at 72°C. Amplification of algC was also verified with native plasmids as PCR template. PCR assays were performed in an ep-gradient PCR (Eppendorf, Germany); products were separated on agarose gels stained with ethidium bromide and documented in a gel documentation system (Alpha Imager HP, Alpha Innotech, USA). DNase-, RNase-, Protease-free water was used as negative control. All PCR chemicals and reagents were purchased from Sigma unless stated otherwise.