Figure 2

Probe based Real-Time PCR analysis of conditional gene deletion. A Strategy for probe and primer placement: The probe (UP #69, Roche Applied Science) is complementary to the loxP site and was used for both reactions. For analysis of the 2lox allele, primers were placed in unique genomic sequence surrounding the 3' loxP site. For the 1lox reaction primers were placed in unique genomic sequence surrounding the loxP site of the truncated allele. B Validation of Real-Time PCR standards using capillary electrophoresis: DNA fragments containing the 1lox and 2lox target sequence respectively were mixed to generate standards with molar ratios ranging from 10/0 to 0/10. Capillary electrophoresis (measured molarities inscribed in the gel image) confirmed that the standards contained the expected ratios of 1lox and 2lox specific sequences. C Real-Time PCR efficiency is not affected by the presence of the alternative allele: 1lox (left graph) and 2lox (right graph) specific Real-Time PCR reactions were conducted using the mixed standards shown in section B. Both reactions show a linear correlation between Crossing point (Cp) and log concentration values indicating that the reaction efficiency is not affected by the presence of the other allele.