Proper integration of the pSN054-1437000-3xHA vector was confirmed by Southern blotting. Genomic DNA from the parent and edited lines was isolated (QIAamp DNA Blood Miniprep Kit), and 10 μg of each was digested with HinDIII, separated overnight on a 0.7% agarose gel and transferred to nylon (Nytran SuPerCharge TurboBlotter, 0.45 μm, GE Healthcare) overnight. The blot was then probed with the left homologous region (PCR product of 14APT-1/14APT-2) labelled with alkaline phosphatase (Amersham AlkPhos Direct Labeling Kit; GE Healthcare) in hybridization buffer (Amersham) at 55°C overnight, washed twice each in primary wash buffer (120 g/l urea, 1 g/l SDS, 100 ml/l 0.5 M sodium phosphate pH 7, 8.7 g/l NaCl, 2 g/l Amersham blocking reagent) and secondary wash buffer (6.05 g/l Tris base, 5.6 g/l NaCl, 2 ml/l 1 M MgCl2, pH 10), then detected with Amersham CDP-Star Detection Reagent (GE Healthcare) and exposed to blue autoradiography film (MidSci, BX810) overnight.
Additional validation by western blotting was done exactly as described in Polino et al. (2020) (link) (section ‘Validation of PMVAPT line’).
Additional validation by western blotting was done exactly as described in Polino et al. (2020) (link) (section ‘Validation of PMVAPT line’).
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