Genejet pcr gel extraction kit

Random mutagenesis of the hdp1 and hdp2 genes was performed with the GeneMorph II Random Mutagenesis Kit (Agilent) using the recommended conditions for obtaining a mean mutation rate of 1 mutation per PCR product. The resulting PCR product was used as a megaprimer for whole-plasmid PCR using Phusion DNA polymerase (Thermo Fisher) (Sarkar and Sommer, 1990 (link)), which was then purified using the GeneJET PCR Gel Extraction Kit (Thermo Fisher) and digested with DpnI (Thermo Fisher) for 1 hr at 37°C to remove the original template plasmid. After digestion, the PCR product was purified again and transformed into electrocompetent DA57390 cells (Supplementary file 1c), which were plated on MacConkey (BD Difco) plates supplemented with 10% lactose and 50 μg/ml of ampicillin and incubated overnight at 37°C. White colonies indicated loss-of-function mutations in the mutagenized genes. These colonies were inoculated into the fresh LB medium supplemented with 50 μg/ml ampicillin and grown overnight at 37°C with shaking at 200 rpm. The plasmids were purified using the EZNA Plasmid Mini Kit (Omega Bio-Tek) according to the manufacturer’s protocol. The nucleotide substitutions were identified by Sanger sequencing.