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Single integration into the expected locus was found in all nine Mosdi1 ReGFP mutants, and expected size bands were obtained in both Mosdi1 ReGFP mutants and wild type strain Guy11.
In addition, the eGFP in invasive hyphae also displayed equivalent fluorescent signal in all five Mosdi1 ReGFP mutants (Fig. 3), indicating that the pMoC-eGFP was integrated into expected locus, with stable expression of eGFP in cytoplasm of M. oryzae.
We examined in detail the genomic region around the MATN3 genes, as the expected locus of the syndecan-1 gene.
All had the crtS gene expression cassettes at the expected locus, as confirmed by PCR analyses with a comprehensive set of primers.
With each fragment, the transposon was found in the expected locus in the genome, confirming that targeting proceeded accurately via homologous recombination.
Four TUs (TUs 3, 8, 12 and 15) yielded non-specific RTPCR products which did not map to the expected locus after sequencing.
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Plasmid integration at the expected loci was confirmed by PCR.
Mapping of both hippocampus weight and coat color in an AIL population using GRAIP returned expected loci.
For coat color, where only two loci are segregating between the parental strains, novel loci would have been indicative of a serious flaw of the method to discriminate false positives from true positives, but only the expected loci were returned.
Complete plastid genome sequences of Cuscuta obtusiflora, Cuscuta exaltata, and Ipomoea purpurea were used to design primers for this study, assess presence of non-group IIA introns within Cuscuta, to eliminate the possibility of gene transpositions in cases of PCR-detected intron and matK loss, and to verify the presence of only the expected loci for genes examined in this study.
A similar loss of expected loci has recently been reported for conserved microRNAs (Fromm et al. 2013).
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