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. 2019 Feb 12;12(2):dmm036863. doi: 10.1242/dmm.036863

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© 2019. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — Validation of ddPCR assay for detection of Kras^Lox-G12D allele using LNA-2 primers. (A-C) One-dimensional droplet plot of LNA-2 assay (comprising probe LNA-2 and primers flanking the recombined LoxP sequence in Kras^Lox-G12D) with Kras^+/Lox-G12D MEF genomic DNA (A), Kras^+/LSL-G12D MEF genomic DNA (B) and Kras^+/+ MEF genomic DNA (C). Primers were annealed at 62°C, following a gradient PCR experiment to determine the optimum annealing temperature (T_a, not shown). A manual threshold of 1000 fluorescence units was selected. (D) Kras^+/Lox-G12D MEF genomic DNA was serially diluted into a background of Kras^+/LSL-G12D MEF genomic DNA. A one-dimensional droplet plot for the 133-bp LNA-2 assay at each serial dilution is shown. A manual threshold of 1100 fluorescence units was selected. (E) Actual versus theoretical copy number of Kras^Lox-G12D at each serial dilution.