PCR optimization for amplification of apoptosis-linked gene 2 (ALG-2) and 18s rRNA gene of Red claw crayfish, Cherax quadricarinatus

The purpose of this research is to optimize the PCR conditions for amplification of ALG-2 and 18S rRNA of red claw, Cherax quadricarinatus. The primers for ALG-2 were designed based on partial mRNA deposited in GenBank with
accession number of JF284559. However, primers for 18S rRNA were purchased according to the sequence reported in Fang et al. (2012). A range of annealing temperatures was tested for 18S rRNA and ALG-2. At the end of this study, the annealing temperature for 18S rRNA is successfully optimized at range of 50 to 62°C. in further, with optimum annealing temperature (53“ C was chosen), 18S rRNA was amplified with different number of cycles ranging from 20 to 40 cycles with two cycles interval. The cycle number that produces half maximal of amplification for 18S rRNA is 28 cycles. Meanwhile, annealing temperature optimization for ALG-2 is partially successful as undesirable PCR products were observed. Thus, the optimum annealing temperature is suggested either 50 or 56°C where specific PCR products were appeared. The results from this work provide optimized PCR conditions for amplification of ALG-2 and 18S rRNA for future gene expression study. In addition, this study can be used as reference for further PCR optimization in order to amplify ALG-2 specifically. Identification of ALG-2 PCR cycle that produces half maximal of PCR amplification is also suggested to be conducted in the future study.