DNA Extraction and Restriction Analysis

H₂O was added to the DNA samples taken during the purification process to a final volume of 100μL. 1ng of plasmid K71 was also added to every sample as a spike-in to normalize for different DNA extraction efficiencies. 100μL of IRN buffer (50mM Tris-HCl pH8, 20mM EDTA, 0.5M NaCl, 0.5% SDS, 10μL Proteinase K (10 mg/mL) were added together with 1μL of RNAse A (10 mg/mL), followed by a 1h incubation step at 37°C. Subsequently, 200μL Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) was added, followed by 2 × 10sec thorough vortexing. The solution was centrifuged for 5 min at 16.000g. The supernatant was transferred to a fresh 1.5mL tube containing 600μL of ethanol and 1.5μL glycogen (10 mg/mL). The tube was left at −20°C overnight. Next, the solution was centrifuged with 16.000 g at 4°C for 30min. The supernatant was discarded and 150μL of 70% ethanol was added to the pellet. After another centrifugation step with 16.000 g at 4°C for 10min, the supernatant was discarded and the DNA pellet dried at room temperature for 10min. The dried pellet was then resuspended in 50μL H₂O. For further analysis, a restriction digestion was performed to analyze the DNA samples in subsequent qPCR reactions. The restriction enzymes used for linearizing the circular DNA were HpaI (ARS305+/−3), BbsI (ARS313+/−3), NcoI (ARS315+/−3) and HpaI (ARS316+/−3). qPCR analysis was performed using the following primer pairs: ARS305: 0463/0466; ARS313: 0552/0553; ARS315: 0970/0971; ARS316: 0837/0838. Primers 0137 and 0138 are used to detect the K71 spike-in and primers 0301 and 0302 were used to detect the unrelated genomic PDC1 locus.