An Optimized CTAB Method for Genomic DNA Extraction from Freshly-picked Pinnae of Fern, Adiantum capillus-veneris L.
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Author:
Date:
2018-07-05
[Abstract] As the sister clade of seed plants, ferns are significant materials for plant phylogeny research. However, the genomic DNA extraction protocol for fern samples like modified CTAB method still lacks robustness. Here, we found that the amount and condition of the pinnae samples are critical for gDNA extraction in fern, Adiantum capillus-veneris L. In 500 μl CTAB solution, the recommended amount of pinnae is about 10-20 mg (2-3 pieces). The condition of the pinnae must be instantly-picked from a plant cultivated in a suitable environment. With these factors under control, it is highly reproducible to get the high-quality gDNA with low degradation rate
[摘要] 作为种子植物的姐妹分支,蕨类植物是植物系统发育研究的重要材料。 然而,像改良CTAB方法的蕨类样品的基因组DNA提取方案仍然缺乏稳健性。 在这里,我们发现羽片样品的数量和条件对于蕨类植物, Adiantum capillus-veneris L中的gDNA提取至关重要。在500μlCTAB溶液中,推荐的羽片量约为10-20 mg(2-3片)。 必须从在合适环境中栽培的植物中立即采摘羽片的状况。 在控制这些因素的情况下,获得具有低降解率的高质量gDNA具有高度可重复性
【背景】CTAB方法已应用于 Adiantum capillus-veneris L的gDNA提取(Han et al 。,2012; Li et al 。,2017年)。 然而,该方案在我们的实验中仍显示出不稳定性和高降解率。 蕨类植物的羽片积聚了大量的次级代谢产物,如多糖和多酚,这对DNA提取不利(Ponnusamy et al。,2015)。
为了提高gDNA提取的稳健性,我们基本上从两个方面对协议进行了优化:1)减少材料用量,仅使用2-3个羽片(约10-20毫克)。 2)必须从在适宜的培养环境(温度:25℃,湿度:65%,16小时白光/ 8小时黑暗处理)中培养的植物中新鲜采摘羽片,并立即用于DNA提取。 ...
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Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells
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Author:
Date:
2018-05-05
[Abstract] Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution ...
[摘要] 骨髓造血干细胞(HSC)需要骨髓微环境来维持和增殖。 骨髓间充质基质细胞(MSC)的培养为体外HSC存活提供适当的环境信号。 在这里,我们提供了描述MSCs培养条件的详细方案,从小鼠骨髓中流式细胞术分离HSCs,并进行称为鹅卵石区域形成细胞(CAFC)分析的MSC和HSC的共培养。 总而言之,CAFC分析可用作高通量体外筛选模型,其中努力了解和开发复杂骨髓疾病的治疗方法。 该方案需要培养MSC,分离LSK细胞(HSC)和执行有限稀释CAFC测定3至4周。
【背景】HSC的增殖,存活和分化潜力非常依赖于其微环境,也被称为小生境。骨髓MSC支持HSC以使其在骨髓龛中保持静止状态。由生态位接收的内在和外在信号有助于将HSC分化为也称为造血的成熟血细胞谱系,而不诱导异常扩增(Yoshihara等人,2007; Spindler等人 ,2014; Hu等人,2016)。鹅卵石区域形成细胞试验(CAFC试验)是长期骨髓HSC和MSC的体外共培养试验。当培养MSC在组织培养皿中完成融合时,将HSC铺在MSC上(de Haan和Ploemacher,2002)。 CAFC测定与骨髓的体内研究相当,并且可用作快速筛选测定以测试HSC的干细胞活性和MSC的支持活性(Ploemacher等人, ...
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