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. 2016 Jul 5;7:1009. doi: 10.3389/fmicb.2016.01009

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Copyright © 2016 Salvadori, Junges, Morrison and Petersen.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Gel analysis of segregants from candidate mutant-containing clones. Primer pair 1 (FP1169/FP1170) amplifies a 346 bp fragment in the parent and a 277 bp fragment in the mutant. Primer pair 2 (FP1171/FP1172) amplifies a 399 bp fragment in the parent but produces no band in the mutant, since primer 1171 is located in the deleted locus. DNA 1 and 4 were extracted from two pure mutant colonies retrieved from the first screening procedure (C1 and C4 in Figure 10). Colonies 1 and 4 (C1, C4) were isolated and grown from the first screening procedure for several generations in order to promote segregation of the mutant strain. L, DNA ladder Gene Ruler 1kb plus (GenScript); DNA control, control with type strain DNA; DNA, extracted DNA after the first screening followed by the colony number; 2C, tested colony after growing for several generations to allow segregation, followed by the colony number.