E coli dh5 alpha competent cells

Recipient sheep were euthanized and fetuses collected at day 75 or 120 of gestation. Samples collected from different tissues were used for genomic DNA extraction using the DNeasy Blood & Tissue extraction kit (QIAGEN). PCR was performed using GoTaq Hot Start Green Master Mix (Promega) using the same primers and conditions described above. PCR products were purified, cloned into pCR^™TOPO^®TA vector (Life Technologies) and transformed into E. Coli DH5-alpha competent cells (NEB). Ten colonies were picked, cultured in LB broth, and plasmid DNA was extracted using a Miniprep kit (QIAGEN). Fast digest EcoRI (Thermo Scientific) was used to identify the positive colonies and samples were sent for Sanger sequencing (Quintarabio). Sequencing analysis was performed as described above.

The pUB26-NUCM plasmid was provided by Prof. U. Brandt (Radboud Institute for Molecular Life Sciences). Site-directed mutagenesis was carried out by PCR using Q5 polymerase (New England Biolabs) with nonoverlapping primers. The linear products were 5′-phosphorylated, blunt-end-ligated and transformed into E. coli DH5-Alpha competent cells (New England Biolabs) for sequencing. Plasmid transformations were performed as described previously (60 (link)). In brief, Δnucm cells (provided by Prof. U. Brandt) were grown at 30 °C in 2xYPD overnight, collected and kept on ice before being washed three times in ice-cold buffer containing 10 mM Tris-HCl (pH 7.5), 0.6 M sorbitol, 150 mM Li-acetate. Approximately 1 μg of plasmid DNA was added to 200 μl of cells and electroporated with a single pulse at 1.5 kV, 200 Ω, and 25 μF (pulse length ∼4.6 ms). Cells were incubated at 30 °C for 2 to 3 h, then plated onto YPD containing 150 μg ml⁻¹ hygromycin B, and incubated for 3 days. Single transformant colonies were selected, confirmed by sequencing, and grown in 2xYPD with hygromycin B. Cell stocks were frozen in 50% (v/v) glycerol.