The influence of the substrate temperature on the preparation of DNA films for studies under vacuum conditions
The influence of the substrate temperature on the preparation of DNA films for studies under vacuum conditions
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真空条件下基底温度对研究用DNA薄膜制备的影响
DOI：
10.1140/epjd/e2011-10510-1
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
David J. Field
中科院分区：
文献类型：
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作者：
M. Śmiałek;Nykola C Jones;R. Balog;N. J. Mason;David J. Field
抽象的
进行实验以确定超螺旋 DNA 膜的物理形式对基底初始温度的依赖性。此类薄膜通常用于涉及低能粒子（如电子或光子）的辐照实验。为了获得此类实验的横截面的绝对值，必须很好地估计薄膜中样品的空间分布。这些研究的目的是将 DNA 薄膜的大小和形状与基底的初始温度相关联，液体样品在抽空之前沉积在基底上。根据我们之前的研究，我们得出结论，为了在真空条件下保存超螺旋质粒 DNA 形式，需要存在防止 DNA 结构水蒸发的试剂。因此，我们研究了由悬浮在含有氢氧化镁、Tris-Cl缓冲液和不同浓度氢氧化钠的溶液中的质粒制备的薄膜的温度依赖性。
为了使胶片可视化，我们使用了带有 CCD 相机的传统光学显微镜和扫描电子显微镜。结果表明，基底温度对薄膜覆盖的基底面积以及 DNA 分子的空间分布都有显着影响。增加在真空下稳定超螺旋 DNA 的氢氧化钠的量会增加 DNA 聚集。经过这些研究，我们得出结论，对于非原子级平坦的基板，产生均匀薄膜的最佳基板温度应在 –5 °C 至 -10 °C 之间，而对于原子级平坦的基板，则应高于 0 °C。
Abstract
Experiments were carried out to determine the dependence of the physical form of supercoiled DNA films on the initial temperature of the substrate. Such films are often used in irradiation experiments involving low energy particles, like electrons or photons. In order to obtain absolute values for cross sections for such experiments, the spatial distribution of the sample in the film has to be well estimated. These investigations aim to correlate the size and form of a DNA film with the initial temperature of the substrate, on which the liquid sample is deposited prior to evacuation. From our previous studies we concluded that the presence of agents preventing DNA structural water evaporation is required in order to preserve the supercoiled plasmid DNA form under vacuum conditions. Therefore, we examined the temperature dependence on films prepared from plasmid suspended in solutions containing magnesium hydroxide, Tris-Cl buffer and various concentrations of sodium hydroxide.
To visualise the films we used a conventional light microscope with a CCD camera and a scanning electron microscope. The results revealed a significant influence of the temperature of the substrate on both the area of the substrate covered by the film as well as on the spatial distribution of DNA molecules. An increase in the amount of sodium hydroxide that stabilises supercoiled DNA under vacuum increases DNA aggregation. After these investigations we conclude that the best temperature of the substrate to produce uniform and thin films should be between –5 °C and -10 °C for substrates which are not atomically flat and above 0 °C for atomically flat substrates.