Quick change mutagenesis kit

pcDNA6A-Myc-EGFR was a gift from Mien-Chie Hung (Addgene plasmid #42665) [48 (link)]. The catalytically inactive PHD2 H374A variant was generated from the pcDNA3.1-PHD2-V5-6xHis backbone via site directed mutagenesis (QuickChange mutagenesis kit, Promega) and was described previously [49 (link), 50 (link)]. PHD2 deletion mutants lacking amino acids 1-139 (PHD2 Δ1-139) and 208-426 (PHD2 Δ208-426) were generated with site directed mutagenesis of pcDNA3.1-PHD2-V5-6xHis creating additional BamHI and XbaI restriction sites in the coding sequence of EGLN1 (PHD2), respectively. Primers were as follows: PHD2 Δ1-139 forward 5′-gggctcggcggtggatcccgacgccatgcccggcaaggagg-3′, PHD2 Δ1-139 reverse 5′-cctccttgccgggcatggcgtcgggatccaccgccgagccc-3′, PHD2 Δ208-426 forward 5′-gcacggcatctgtctagaggacgacttcctcggc-3′, PHD2 Δ208-426 reverse 5′-gccgaggaagtcgtcctctagacagatgccgtgc-3′. Afterwards, mutants were digested with BamHI (for PHD2 Δ1-139) and XbaI (for PHD2 Δ208-426), purified (Gel/PCR DNA fragments extraction kit, GeneAid), re-ligated, and transformed into XL-1 blue competent cells. Several clones were picked for plasmid propagation and restriction analysis. All constructs were verified by DNA sequencing.