Dna rna duet kit miniprep kit

All animal experiments were carried out under and in accordance with a UK Home Office Project Licence in a Home Office-designated facility. Primordial and gonadal germ cells were isolated from embryos obtained from a 129Sv female and GOF18ΔPE-EGFP 36 male cross. Genital ridges from 1-2 litters (7-8 embryos per litter) were dissected out and digested at 37°C for 3 min using TrypLE Express (Thermo Fisher Scientific). For E9.5 and E10.5 stages, posterior half of the embryo was dissected out and digested with trypsin solution (0.25% trypsin, 10% chick serum (both from Life Technologies), 1.27mM EDTA (Sigma)). Litters were pooled for stages up to and including E11.5 to obtain more material. Enzymatic digestion was neutralized with DMEM/F-12 (Gibco) supplemented with 15% fetal bovine serum (Gibco) after manual dissociation by gentle pipetting. The cells were spun down by centrifugation and resuspended in 0.1% BSA PBS. GFP-positive cells were isolated using an Aria Fusion (BD Bioscience) flow cytometer and sorted into ice-cold PBS. Total RNA was isolated from using DNA/RNA Duet Kit miniprep kit (Zymo Research, USA).