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. 2018 Jul 30;8:11392. doi: 10.1038/s41598-018-29621-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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EvaGreen duplex reactions do not affect calculated template concentration. (A) Positive droplet intensity as a function of primer concentration and amplicon length. Amplicon lengths are 69 (BA), 137 (ND3), 144 (ND1) and 83 (DL2). Inset: equation 3, k fitted as a square law function of amplicon length (L). (B,C) Independent singleplex and multiplex reactions of DL2 and BA demonstrate 100 nM of each primer provides effective separation of droplet clusters in both 1D and 2D. E&F. Independent singleplex and multiplex reactions of DL2 (100 nM) and ND3 (40 nM). (D,G) Copy number for singleplex (n = 3) and multiplex (n = 3) reactions for both assays. (B,C,E,F) EvaGreen intensity collected on QX200 filters suited for FAM (green) and HEX (orange) dyes.