E coli dh5α

To generate UAS-mtAcp1 transgenic fly line, cDNA clone for mtACP1RB (DGRC: AT22870, Cat# 11961) was digested with EcoRI and XhoI (NEB; Cat# R0101 and Cat# R0146 respectively) in NEBuffer 2.1 (NEB, Cat# B7202) for two hours at 37°C. The 783bp insert was eluted from 1% agarose (Sigma-Aldrich, Cat# A9539) gel using the GeneJET Gel Extraction Kit (Thermo Scientific, Cat# K0692). Separately, the pUAST vector (DGRC, Cat# 1000) was digested with EcoRI and XhoI in NEBuffer 2.1 (NEB, Cat# B7202) for two hours at 37°C and the digested DNA was eluted from 1% agarose gel by using the GeneJET Gel Extraction Kit. Ligation of the insert (783bp) DNA with the digested pUAST vector was performed using T4DNA ligase (NEB, Cat# M0202) in the buffer provided, overnight at 16°C. The ligated product of 9.5Kb was eluted from 1% agarose gel and purified. E. coli DH5α (MTCC, Cat# 484) competent cells were then transformed with the ligated product using the conventional heat shock method and colonies were selected for ampicillin (HIMEDIA, Cat# TC021) resistance. Plasmid DNA were isolated from the transformed colonies and dissolved in Tris-EDTA buffer (pH 8). The clone was then sent to the fly facility of Centre for Cellular and Molecular Platforms (C-CAMP), Bangalore for the generation of the UAS-mtAcp1 transgenic line.