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An array of 85 negative controls indicated that low template samples are unlikely to result from contamination by ambient exogenous DNA.
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Furthermore, "no reverse transcription" and "no template" samples were included as negative controls for each reaction.
The target gene names, RefSeq numbers and primer sequences are shown in Table 4. Triplicate cDNA template samples were amplified and analyzed in the Prism 7000 sequence detection system (Applied Biosystems).
Triplicate cDNA template samples were amplified and analyzed.
Wild type and no template samples were added in each assay as positive and negative controls.
The 200 template samples were sequenced on 318 chips using the PGM platform with an Ion PGM 200 sequencing kit (Life Technologies Corporation).
Quadruplicate (n = 23) and duplicate (n = 2; with limiting amount of RNA for cDNA synthesis) cDNA template samples were amplified and analysed on the ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA).
Quadruplicate (n = 38) and duplicate (n = 3; with limiting amount of RNA for cDNA synthesis) cDNA template samples were amplified and analysed on the ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA).
Eight indexed DNA template samples were pooled at equimolar ratio and sequenced in one lane of the flow cells of HiSeq2000 (Illumina, San Diego, CA) with single-end, 75-base read lengths.
DNA contamination within the RNA template sample was checked either by use of no RT controls or using primers spanning introns.
The 200 ng gDNA template sample was amplified separately after evaluation of the genotyping performance of the wgaDNA produced from 1 – 100 ng gDNA.
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