RNA提取实验

READ THROUGH ALL CAUTIONS BEFORE TRYING THIS EXPERIMENT Materials •Rat liver (fasted rat) •Liquid nitrogen •p-Amino-salicic acid •Phenol mixture •Homogenizer or blender6 &bull ...

1）检测RNA 溶液的吸光度 280、320、230、260nm下的吸光度分别代表了核酸、背景（溶液浑浊度）、盐浓度和蛋白等有机物的值。一般的，我们只看OD260/OD280（Ratio，R）。1.82.0时，我们认为RNA 中蛋白或者时其他有机物的污染是可以容忍的，不过要注意，当你用Tris作为缓冲液检测吸光度时，R值可能会大于2（一般应该是<2.2的）。当R<1.8时，溶液中蛋白或 ...

This is the preferred method for yeast RNA preparation use Gloves and RNAse free solutions throughout. 1. Use a YPD overnight culture to innoculate fresh YPD media and grow cells at 30 degrees overni ...

T℃OS-M6 cells grow in 6-well plates 2ml media total T℃V1PD cells grow in 100mm plates. 1: Aspirate off the media from the cells and was the cell monolayers with 2x5ml (To 100mm dish) or 2x1ml (to 6-w ...

Based on: Wan C.-Y. and Wilkins T.A. 1994. Anal. Bioch. 223:7-12. 1.Collect young expanding leaves (or other tissue) and freeze in liquid N2 and store at -80℃. 2.Pulverize tissue to a fine pow ...

Preparation of Cells 1.Prepare or collect between 2x107 cells for each mRNA prep (will yield about 10-20µg of mRNA). If PBMCs from a whole blood sample are to be used the sample should be prepar ...

RNA Isolation - Volumes and weights are for 10 ml cultures (1-2 x 107 cells/ml). 1.Spin down cells decant and resuspend in 0.2 ml extraction buffer with SDS and transfer to a 1.5 ml eppendorf tu ...

真核细胞的mRNA分子最显著的结构特征是具有5’端帽子结构（m7 G）和3’端的Poly(A)尾巴。绝大多数哺乳类动物细胞mRNA的3’端存在20-30个腺苷酸组成的Poly（A）尾，通常用Poly（A+ ）表示。这种结构为真核mRNA的提取，提供了极为方便的选择性标志，寡聚（dT）纤维素或寡聚（U）琼脂糖亲合层析分离纯化mRNA的理论基础就在于此。 mRNA的 ...

第一节 概 述 从真核生物的组织或细胞中提取mRNA，通过酶促反应逆转录合成cDNA的第一链和第二链，将双链cDNA和载体连接，然后转化扩增， 即可获得cDNA文库，构建的cDNA文库可用于真核生物基因的结构、表达和调控的分析;比较cDNA和相应基因组DNA序列差异可确定内含子存在和了解转录后加工等一系列问题。总之cDNA的合成和克隆已成为当今真核分子生物学的基本手段。自70年代中叶首例cDNA克 ...

1) Harvest 1x105 to 108 cells. Wash 1x w/PBS and freeze in liquid N2 and store @ -70℃ 2) Resuspend each pellet in 1.5ml lysis buffer (300μl 5x Lysis 75μl 200mM VR 1.125ml H2 O 3) Incubate on ic ...

Prior to Extractions ・ Bake all necessary glassware metal spatulas mortars and pestles overnight in a 200°C oven after wrapping them in aluminum foil. For each sample you should minima ...

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RNA for S1 or PE analysis must be free of chromosomal DNA. This can be accomplished by extraction of the RNA with hot phenol but phenol can especially if its pH is acidic cause the specific loss ...

AMES/Chloroform extraction in our lab to extract total from potato leaves for viroid analysis. The protocol is as follows: AMES Buffer (for 200mL): 11.7g NaCl 160 mL dH2O &nbs ...

All solutions used in RNA preparation should be treated with DEPC as follows: add DEPC as follows: add DEPC to 0.1% and vigorously stir for one hour to O/N followed by autoclaving for 30 minutes ...

1. Snap freeze ~107 cells or ~100 mg of tissue in liquid nitrogen or dry ice/ethanol. 2. Transfer the frozen sample to a mortar and pestle and grind to a fine powder. Or use a hand-held tissue grinder ...

In the past poly(A)+ RNA has not been detected in prokaryotes. In the early 80s a new method of isolating RNA from bacteria was developed involving lysis by protease K in the presence of SDS and ...

CONCERT细胞质RNA 纯化试剂是一种新颖的单相酚溶液，是专门设计用来从新鲜或冷冻的培养的动物细胞 或组织中快速、简单地纯化高质量的细胞质RNA 。 应用 利用CONCERT™细胞 质RNA 试剂分离的总RNA 能够用于构建cDNA文库，RT－PCR，northern印迹分析，斑点印迹杂交，poly（A＋）筛选，体外翻译，RNA 酶保护，和分子克隆。 现在所有的商品化试剂不仅分离细 ...

Hot phenol may also be used to remove DNA. 1. Add equal volume of TE-saturated phenol to RNA solution. Mix. 2. Heat to 70 ℃ for 5 minutes. 3. Centrifuge at top speed for 10 minutes at room temperature ...