Gotaq g2 flexy dna polymerase

Lonp1^wt/– mice were generated with a knock-out allele encompassing exons 5 and 8 from Biogem Srl (Ariano Irpino, Italy) on a C57BL/6 background^{27 (link)}. The genotyping was performed on genomic DNA extracted from ear samples and obtained using the commercial Easy-DNA™ gDNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). 20–50 ng of DNA were amplified in 25 μL of a reaction mix containing 1X GoTaq Flexy Buffer (Promega Corporation, Madison, WI, USA), 2 mM MgCl₂, 0.2 mM of each dNTP (Promega), 0.03 U of GoTaq G2 Flexy DNA Polymerase and 0.2 μM of the following primers: forward 5-CAGGGAAGAAACTGAAGTCAGGC-3, reverse 5-CACTCTGGTTCATGGCCACC-3. A 925 bp PCR product was obtained only for knock-out allele. All mice were housed in ventilated cages for a 12-h day/night cycle with access to water and food ad libitum. All animal experiments and protocols were performed in accordance with ARRIVE guidelines and approved by the Italian law protecting animals used for scientific purposes (authorization n°253/2017-PR, released on March, 21st, 2017 from Italian Ministry of Health).

The Lonp1^wt/− mouse (background C57BL/6) was provided by Biogem Srl (Ariano Irpino, Italy) by generating a knock-out allele encompassing exons 5 and 8 (Figure S1). Genotyping was performed by polymerase chain reaction (PCR) on 20–50 ng of genomic DNA obtained from ear samples using the commercial Easy-DNA™ gDNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) amplified in 25 μL of reaction mixture having this composition: 1X GoTaq Flexy Buffer (Promega Corporation, Madison, WI, USA), 2 mM MgCl₂, 0.2 mM of each dNTP (Promega Corporation), 0.03 U of GoTaq G2 Flexy DNA Polymerase and 0.2 μM of each primer. The cycling parameter protocol included an initial step of denaturation at 95 °C for 1 min followed by: 2 cycles at 95 °C 15 s, 64 °C 15 s, 72 °C 1 min; 2 cycles at 95 °C 15 s, 61 °C 15 s, 72 °C 1 min; 20 cycles at 95 °C 15 s, 58 °C 15 s, 72 °C 1 min; 10 cycles at 95 °C 15 s, 55 °C 15 s, 72 °C 1 min and a final extension step at 72 °C for 10 min. The following primers were used: forward 5′-cagggaagaaactgaagtcaggc-3′, reverse 5′-cactctggttcatggccacc-3′, producing a 925 bp product only for knock-out allele, detected by agarose gel electrophoresis.