Genotyping by PCR
Mouse tail DNA was isolated and purified by cutting approximately 0.5 cm of mouse tail and digesting it in 500 μl lysis buffer (50 mM Tris-HCl, pH8.0, 100 mM EDTA, 125 mM NaCl, 1% SDS, 200 μg Proteinase K) with rocking overnight at 55°C. 200 μl saturated NaCl was added into digested solution with vigorous shaking for at least 60 seconds. Mixed solution was spun down at maximum speed in top micro-centrifuge for 20 min. The supernatant was transferred to a new tube with equal volume 100% EtOH and mixed by inversion. The DNA pellet was washed in 70% EtOH and resuspended in 100–150 μl TE buffer. UV spectrophotometry and electrophoresis were used to analyze the quality and quantity of the genomic DNA. Genotyping was performed by multiplex PCR reaction using three separate primers (two Abcg8 gene specific primers, mg8-in3F 5'-CCCAATGATGAATGAGACGCT-3', mg8-R9 5'-TTTGTCACGCTGGGCCTGG-3' and neoF for identification of Abcg8 target status). The PCR reaction consisted of 1x PCR buffer (1.5 mM MgCl2, 16 mM (NH4)2SO4, 0.1 mM dNTPs, 67 mM Tris-HCl (pH8.8)) 1 μM of each primer, water and 1.0 units of Taq polymerase. The reactions were cycled as follows: 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min for 35 cycles. The PCR products were identified by electrophoresis.