High efficiency 5 alpha competent e coli cells

The hda-1 3 R RNAi construct was designed to target a unique 774 bp region in the 3 UTR of hda-1. This region in hda-1 was amplified from N2 genomic DNA with primers containing restriction digest sites for EcoO109I and SacI-HF restriction enzymes. Purified PCR products and purified DNA from miniprep of the L4440 vector were digested with EcoO109I and SacI-HF in rCutSmart^™ (NEB) buffer for 1 h or 16 h at 37 °C. The digested products were purified and ligated together using T4 DNA ligase (NEB). The ligation product was then retransformed into high-efficiency 5-alpha competent E. coli cells (NEB) which were grown overnight at 37 °C. Colonies were grown for 12–16 h overnight at 37 °C in LB containing 100 μg/mL carbenicillin and plasmids were purified by miniprep and verified with Sanger sequencing. 1 μl of the correct plasmid was then retransformed into homemade HT115 competent cells and used for RNAi. See Supplementary Information - Supplementary Table 2 for primer sequences.

The hda-1 3′ UTR RNAi construct was designed to target a unique 774 bp region in the 3′ UTR of hda-1. This region in hda-1 was amplified from N2 genomic DNA with primers containing restriction digest sites for EcoO109I and SacI-HF restriction enzymes. Purified PCR products and purified DNA from miniprep of the L4440 vector were digested with EcoO109I and SacI-HF in rCutSmart^TM (NEB) buffer for 1 h or 16 h at 37 °C. The digested products were purified and ligated together using T4 DNA ligase (NEB). The ligation product was then retransformed into high-efficiency 5-alpha competent E. coli cells (NEB) which were grown overnight at 37 °C. Colonies were grown for 12–16 h overnight at 37 °C in LB containing 100 μg/mL carbenicillin and plasmids were purified by miniprep and verified with Sanger sequencing. 1 μl of the correct plasmid was then retransformed into homemade HT115 competent cells and used for RNAi. See Supplementary Data 5 for primer sequences.