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Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5′ upstream region.
Two of the primary pHEX22 transformants (from two events) had a normal phenotype.
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Transformants from five independent transformation experiments (i.e., 33 clones) were tested for their maximal antibiotic resistance.
For each transformation vector, a total of 10 primary T0 transformants from five independent transgenic lines were first screened for the presence of CX3 or CX5 transgene by PCR.
No rescue of the motility defect was detected in over 700 transformants from three independent experiments.
For comparison purposes between the transformants from the two different backgrounds, the growth curve of the parental strain with the empty vector was included in the plates with the BY4741_ haa1Δ transformants.
Five co-transformants obtained from two independent callus lines were identified in tissue culture by PCR using primers specific for the coding regions of the transgenes.
As a second test for stable integration, selected transformants from each four 96-well plates were used to assemble 384-colony arrays on EMM + YC − uracil and EMM + FOA Omni plates (Nalge Nunc International) using a 96-floating-pin replicator and a colony copier (VP409 and VP381, V&P Scientific, Inc).
The eight transformants arose from three independent events: #6, #29, and #35, had 2, 3, and 1 copies of the transgene, respectively (Fig. 5A, lanes 3, 5, and 6).
Co-cultivation of A. tumefaciens with V. albo-atrum conidia on tobacco stems (Fig. 3A) resulted in the isolation of 10 fungal transformants in total from two independent experiments (31 plates in total, 1 stem per plate).
The average increase and standard deviation in adhesion by the lig transformants from at least three separate experiments are reported.
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