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Finally, the beads were counted using a hemocytometer to get a concentration of 106 per ml.
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The PCR-amplified fragments on beads were washed and the bead number was counted using a Coulter Counter Z1 single threshold instrument (Beckman Coulter Co).
The final RR was recorded for the three tools as follows: (1) when using the timer alone, the CHW was asked for the final RR count, (2) when using both the timer and beads, the beads were counted back by a research assistant who provided the final RR count and (3) for the mobile phone application, the result was read from the screen.
Using 1000 and 2000 beads in the assay more than 50 beads were counted resulting in a robust MFI signal.
At least 100 beads were counted per analyte.
Beads classified as 1001 were counted as wild type and 0110 beads were counted as mutants.
Puncta to ring structures showing the proteins directly at the beads were counted as positive localization to beads.
The number of cells with at least one bead was counted after bead labeling and cell enrichment on a magnet (after bead labeling, 1.2 × 10, gray bar, left).
DNA-containing beads were enriched and counted using a CASY Cell Counter DT (Roche Innovatis AG), processed using aXLR70 sequencing kit (454 Life Sciences/Roche Diagnostics), and loaded onto a picotiter plate for pyrosequencing on a 454 Life Sciences Genome Sequencer FLX machine (454 Life Sciences/Roche Diagnostics).
The enriched bead samples were then counted using a Z1 Coulter Counter (Beckmann Coulter, CA, USA) to calculate the percent enrichment (i.e., the percent of initial beads that contained sstcDNA).
Mycobacteria were disaggregated by vigorous vortexing with glass beads and counted using a Neubauer hemocytometer as previously described [ 16].
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