Pcr clean up and gel extraction kit

Insertion and deletion mutations were quantified with the assumption that hepatocytes comprise 60% of the liver^{54 (link)}. Harvested livers were homogenized in saline at a 1:1 ratio and stored at −80 °C until extraction was performed. Genomic DNA was extracted from homogenized liver tissue using the MasterPure Complete DNA and RNA Purification kit (Lucigen) following the manufacturer’s instructions. The region surrounding the Cypor gRNA cut site (Forward primer: 5′-GTTTGCGGGTGTTAGCTCTTC-3′, Reverse primer: 5′-AGTCTACTTCAGTCGCAGCC-3′) and Hpd gRNA cut site (Forward primer: 5′-GAACTGGGATTGGCTAGTGC-3′, Reverse primer: 5′-GCCCTTCCCTACATCCTAGT-3′) were amplified with MyTaq Red Mix (Bioline). PCR products were then purified using the PCR clean-up and gel extraction kit (Macherey-Nagel) and Sanger sequenced. The forward primers served as sequencing primers. Indels for each mouse were analyzed using the TIDE software, https://tide.nki.nl²⁶ .

Plasmids were constructed with Gibson assembly [32 (link)] or classical restriction and ligation. PCR-generated fragments (All-in HiFi, highQu, Kraichtal, Germany) were assembled into isolated plasmids (Plasmid GeneJET Miniprep kit, Thermo Fisher Scientific, Schwerte, Germany) that were linearized by restriction. Oligonucleotides used in this study were obtained from Metabion (Planegg/Steinkirchen, Germany) and are listed in Table 2. Standard reactions like restriction, and PCR were performed as described previously [33 ]. If applicable, PCR products were purified using the PCR clean-up and gel extraction kit (Macherey-Nagel, Düren, Germany). For transformation of E. coli DH5α, the RbCl method was used and C. glutamicum was transformed via electroporation [34 (link)] at 2.5 kV, 200 Ω, and 25 µF. Vector construction was confirmed by sequencing of cloned inserts.