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A BAC library was produced from MSSTATE#1 leaf nuclear DNA according to Peterson et al. [ 61].
For immunodetection, maize leaf nuclear proteins were obtained from nuclei enrichment extracts according to the procedure described by Casati et al. [ 56].
Analysis of a bone marrow sample obtained by aspiration indicated an abnormally high proportion of eosinophils (total eosinophils 14%, metamyelocyte eosinophils, 2%; rod nuclear eosinophils, 2%; and leaf nuclear eosinophils, 10%).
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Note that the chromocenter number versus leaf cell nuclear area scatter plots shown in Figure 6A were generated in a separate experiment from the one shown in Figure 4, but developmentally matched leaf tissues were harvested as described above.
Leafs were grinded, nuclear buffer was added and samples homogenized and filtered on Miracloth layers.
These protocols were successful in amplification of nuclear DNA from leaf and stem tissues using ISSR and SSR markers and also chloroplast DNA amplification using primers for rbcL gene.
For the nuclear staining, tobacco leaf peals were incubated for 10 min with DAPI (1 µg/µl) before observing under fluorescent microscope with FITC filter.
Loss of CRWN4 also reduced nuclear size in leaf cells as was recently shown by Sakamoto and Takagi (2013) [ 7].
Generally, RP mutants share developmental abnormalities such as reduced shoot growth, reduced cell proliferation and increased nuclear ploidy in leaf cells [ 13- 15].
Co-transfection experiments with RFP AIP1 and GFP ABAP1 in Nicotiana benthamiana leaf abaxial epidermis confirmed the nuclear localization of AIP1 [ 4], and showed co-localization with ABAP1 (Fig. 5d).
Combining a crwn1 loss-of-function mutation with a deficiency in any of the remaining three paralogs causes a further reduction in nuclear size in leaf cells.
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