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PCR and a Bioanalyzer were used to check the RNA for DNA contamination, quality, and concentration.
To ensure that the samples were processed correctly and without external contamination, quality control checks involved four positive controls for each assay and four negative controls (no template controls) per plate.
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The occurrence of enterococci in observed areas is significant to determinate of faecal contamination and quality of the water.
The variation across samples in the proportion of matches could be due to numerous factors, including variation in exogenous DNA contamination or quality of the sequence data.
After trimming vector contamination, low quality bases, and eliminating trimmed sequences with length less than 100 bp, three libraries resulted in 58,842 high-quality EST sequences with an average read length of 755±171 bp and an average high quality base pairs per read of 670±181.
Controls are included on each array for genomic DNA contamination, RNA quality and general PCR performance.
Apart from lack of information on and correction for contamination, the quality criteria were mostly fulfilled (see supplementary table 2).
To ensure that there was no genomic DNA contamination, the quality of RNAs was accessed by PCR with primers in one exon.
Raw reads were trimmed for adapter contamination, minimum quality score (20), experimentally introduced cytosines, and minimum length (20bp) using Trim Galore v.0.3.3 [ 35].
Arrays profiled 84 pathway-specific genes with validated primers and contained internal control primers to assess genomic DNA contamination, RNA quality, and PCR amplification efficacy.
After cloned sequences were filtered for vector contamination and quality, we obtained 2144 expressed sequence tags (ESTs; GenBank accession numbers: EG325041– EG325041).
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