Optimization of PCR Condition for amplification of ferritin 3 and 18S rRNA of cherax quadricarinatus.

Iskandar Mohd Zaib, (2016) Optimization of PCR Condition for amplification of ferritin 3 and 18S rRNA of cherax quadricarinatus. [Undergraduate Final Year Project Report] (Submitted)

Abstract

This study presents an optimization method for Polymerase Chain Reaction (PCR) of ferritin 3 gene from redclaw crayfish, Cherax quadricarinatus. The sample was taken from the hepatopancreas of the crayfish. Specific primer was designed based on the ferritin 3 and 18S rRNA gene (partial mRNA) deposited in GenBank. The sequence of the ferritin 3 then deposited in the primer design program, Primer3 to obtain specific primer, and 18S rRNA used as control. A range of annealing temperature was tested for ferritin with‘ ferritin 3 melting temperature as references. The annealing temperature for ferritin 3 is optimized at temperature range of 45 ⁰C to 57 ⁰C. The ferritin 3 is best optimized at annealing temperature of 45 ⁰C or 51 ⁰C where the specific PCR product was observed. For l8S rRNA, the annealing temperature is at range 50 ⁰C to 62 ⁰C . These results provide optimized PCR condition for amplification of ferritin 3 and 18S rRNA for future gene expression study. Further optimization of ferritin 3 and 18S rRNA in terms of number of cycle suggested to be performed in the future study.

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