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Specific primers were developed for each of the genes found to be duplicated and their respective duplicates (Table S2).
Direct sequencing was performed using all the PCR products obtained from cDNA amplification as template to confirm the specificity of the primers developed for all the genes found to be duplicated and their respective duplicates.
D. virilis 1051.49 (Chaco, Argentina); D. persimilis 14011-0111.48 (California, USA); D. willistoni 14030-0811.16 (Rocha, Uruguay) and D. mojavensis 15081-1352.00 (California, USA) were used to address the expression profile of the different genes found to be duplicated and their respective duplicates.
Total RNA was not pooled but kept as respective duplicates, and samples were sent to the National Center for Genome Resources (NCGR, Santa Fe, NM).
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Then 0.1 mL of each test plasma sample and the two quality control sera was added to the respective duplicate tubes.
Between 100 000 and 200 000 erythrocytes or 10 000–20 000 enriched parasites were analyzed from each sample and its respective duplicate to ensure reliability.
CMs of the tumour cell lines NCI-H460, HCC1143, DU-145 or PA-TU-8902 PA-TU-8902 PA-TU-8902l were applied thawed respective well in duplicands.
A possible explanation for this could be the occurrence of compensatory effects with either direct backups for all genes in the respective isoform via duplicates, which we actually found to be the case for a significant fraction of those isoforms (14%), or alternative protein assemblies that are similar enough to provide the same functionality in the cell.
(For example, the coding we use to identify high-cost patients and subsets of that segment is pages long). Our analysts maintain data integrity across the organization and prevent the respective committees from duplicating efforts.
A specific standard curve was performed in parallel for each gene to assess the specificity of the products, for quantification of the respective transcripts in duplicate.
After each step, the wells were washed three times with 200 μl of wash buffer; 50 μl of dilution buffer (0.1% BSA, 0.05% Tween20, 20 mM Trizma base, 150 mM NaCl, pH 7.3) per well plus 50 μl of each sample or standard dilutions for recombinant human TNF (10,000 to 39 pg/ml; R&D Systems) were then added to the respective wells (in duplicate) and incubated at room temperature for 2 hours.
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