核酸样品制备

现代核酸样品制备方法则是基于高效固相分离,我们提供领先的illustra系列核酸前处理解决方案,用更少的动手时间、提高自动化效率、并将有毒化合物的使用减少到最低水平,相比传统的核酸样品制备方法如酚-氯仿抽提、示差沉淀、乙醇沉淀,或者凝胶分离,效率和质量大大提高。

对方法的选择依赖于核酸的类型、预期的下游应用,以及对产量、质量、纯度和规模的要求。组织通常需要机械性或化学性分散,具体的方法取决于组织类型。细胞裂解步骤非常关键,必须将核酸酶灭活,否则会造成基因组DNA或RNA的降解,但需要尽量避免降解基因组DNA或RNA的核酸酶失活。

许多的工作流程需要在不同步骤间改变缓冲液的条件或浓度。核酸制备可采用传统方法,如乙醇沉淀,或者采用现代的方法,比如乙醇沉淀,或者采用现代的方法,根据片段大小使用预填充凝胶过滤层析介质的柱子进行分离。

细菌细胞可以通过离心方法收集,裂解后,质粒DNA会倾向于结合到膜或固相介质上(如阴离子交换树脂或硅树脂)。在纯化的质粒DNA被洗脱之前,洗涤步骤可以去除污染物,比如碳水化合物和蛋白质。分离出的质粒DNA的产量和纯度,常受细胞培养密度、生长的持续时间、培养基、质粒的类型(髙拷贝数或低拷贝数)、插入片段的大小,以及使用的宿主菌株的影响。

    核酸提取试剂盒通常基于二氧化硅或阴离子交换固相介质,适合自动化,通过离心或真空进行操作。污染的RNA和蛋白质可以用酶去除。另外一个选择是,利用FTA技术,将基因组DNA固定并稳定到一张化学处理过的滤纸卡片上,使蛋白质变性,并在室温条件下长期保存DNA,取样步骤中可实现同步纯化。

    利用全基因组扩增的技术,可以从少至10ng的纯化的DNA中制备得到大量的基因组DNA。

由于纯化方法的改进和下游方法灵敏度的提高,当前应用趋势是纯化总RNA而不是mRNA。一般采用基于溶液或二氧化硅的方法,通常涉及一种离液剂或者其他的化合物以失活RNases,比如,胍盐能够抑制RNases并促进DNA和RNA与二氧化硅的结合,此外DNases用于去除污染的DNA。

多腺苷酰化的mRNA能够利用一种结合有oligo-dT的固相,通过亲和色谱特异地从真核生物中分离出mRNA。

将实验室和试剂中的RNases水平降到最低至关重要。同时,样品也必须被速冻以确保组织中的RNases不会降解RNA,尤其是在表达研究中。

Much of GE Healthcare's filtration media are available in either flat format or in encapsulated formats, such as syringe filters, capsule filters, and filtration microplates.

  • Flat filters: Circles or sheet format. Circles would normally be used in an appropriate filter holder.
  • Prepleated filters (only for cellulose papers): Prefolded/pleated filter paper circles for use in conical filter funnels.
  • Syringe filters: Small, encapsulated, disposable filter units intended for filtration of small sample volumes using a syringe.
  • Syringeless filters/filter vials: Encapsulated, disposable filter units for small volume sample preparation not requiring the use of a syringe to drive the filtration step.
  • Inline filters: Encapsulated, disposable filter units intended for connection to flexible tubing. Units are available for filtration of liquids and for air/gas applications.
  • Capsule filters: Disposable filter units incorporating pleated filter media for larger volumes of liquid and air/gas applications.
  • Filtration microplates: Microplates with incorporated filter media for simultaneous filtration of multiple samples.

 

Filter characteristics and typical properties

The selection of a laboratory filter depends on the conditions and objectives of your experiment or analytical procedure. The three most important characteristics of any laboratory filter are:

  • Particle retention efficiency
  • Fluid flow rate through the filter
  • Loading capacity

According to your particular application, other important filter characteristics may also require examination, for example wet strength, chemical resistance, purity, and ash level. 

Typical properties of filter papers and glass fiber filters

Membrane materials

Chemical compatibility chart of membranes and housings


Filtration Methods for flat filters

  Gravity Vacuum Pressure
Cellulose Filter Papers Suitable Suitable Not generally used
Glass Microfiber Not suitable Suitable Suitable
Membranes Not suitable Suitable Suitable


Maximum practical volumes of cellulose filter paper circle sizes (quadrant folded)

Volume (ml) 15 20 35 75 135 300
Filter Diameter (mm) 90 110 125 150 185 240