Isolation and cloning of ABCB8 gene from Rasbora sarawakensis

Abdul Qawiem, Bin Jumaan. (2015) Isolation and cloning of ABCB8 gene from Rasbora sarawakensis. [Final Year Project Report] (Unpublished)

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Abstract

ATP-binding cassette (ABC) genes encode ABC transporter proteins that have major functions in ATP hydrolysis which assist the take up of nutrient through the plasma membrane. The aim of this research is to identify the gene of ABCB8 gene and subsequently clone the ABCB8 gene into pGEM-T easy vector. Total RNA was primarily isolated from whole Rasbora sarawvakensis that were homogenize using Tri reagent and phenol chloroform precipitation. Afterward, first strand cDNA were produced and the transcript was amplified with PCR using degenerate primers targeting the conserved region of the gene. This generated an approximately 668 bp amplicon. Then, the band was extracted and cloned into pGEM-T easy vector. Transformation using prepared E. coli XL1- Blue generated an efficiency of 10^6 transformants, with 7 of blue and 8 of white colonies. Additionally, 4 white colonies were tested with colony PCR and shows the presence of insert. More verification of insert using Notl restriction digestion was carried out which produced two bands. Then, the plasmid minipreparation product was sent for sequencing and the result was confirmed using BLAST. Thus, sequencing result show an 87% similarity to Danio rerio ABCB8 gene from the genbank. Based on this project, advance temporal and spatial expression of ABCB8 gene should be study more with the purpose of fully utilize the Rasbora saralvakiensis as a model organism for disease study.

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