Embryonic translation extract preparation

Embryonic extract Preparation

C. elegans embryonic pellets were homogenized in hypotonic buffer [10 mM HEPES-KOH pH 7.4, 15 mM KCl, 1.8 mM Mg(OAc)2, 2 mM DTT] with a prechilled Kontes dounce homogenizer. The extract was then centrifuged twice at 13,200 rpm for 10 minutes at 4°C. The supernatant was loaded onto a Column- Prep (BioRad) stacked with Sephadex G-25 Superfine beads (volume of beads was four-times the volume of the supernatant, Amersham Bioscience) and pushed into the matrix with 1 supernatant volume of isotonic buffer (30 mM HEPES-KOH pH 7.4, 100 mM KOAc, 1.8 mM Mg(OAc)2, 2 mM DTT). Multiple elutions (5-7) were gathered and protein concentrations were determined by Bradford. The average concentration of active fractions ranged from 10-30 mg/mL.

In vitro Translation Assays

Reactions were typically set up as follows: each 12.5 ul reaction contained 5 ul embryonic extract, 0.1 mM spermidine, 60 uM amino acids, 36 mM HEPES-KOH (pH 7.4), 2 mM Mg(OAc)2, 65 mM KOAc, 0.1 ug/ul calf liver tRNA, 0.096 U/ul RiboLock RNase Inhibitor (Fermentas), 16.8 mM creatine phosphate, 81.6 ng/ul creatine phosphokinase, 0.8 mM ATP, and 0.2 mM GTP). Reactions were incubated with mRNA (1 nM final) at 17ºC for 0 to 3 hours, as indicated. Luciferase activities were analyzed with the Dual-Luciferase® Reporter Assay System (Promega). To assay for miRNA activity, reactions were pre-incubated with 50 nM (except where indicated) 2’-O-Me oligonucleotides (Dharmacon) prior to mRNA addition for 30 minutes at 17°C. The 2’-O-Me miRNA inhibitors were designed as antisense oligonucleotides to the mature miRNAs according to Wormbase registry (www.wormbase.org).