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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 powder in a ...

RNA extraction buffer:0.1M NaCl2% SDS50mM Tris/HCl (pH9)10mM EDTA20mM ß-mercaptoethanol1. Grind leaf tissue on liquid N in mortar and pestle.2. Transfer the ground tissue to a 10ml conical bottom cent ...

RNA interference (RNAi ) by double stranded RNA (dsRNAs) molecules of approximately 20-25 nucleotides termed short interfering (siRNAs) is a powerful method for preventing the expression of a particul ...

Chemicals needed:Glucose L-( )-Arabinose Sigma Cat # A3256 L-Rhamnose Sigma cat # R3875 Cb (carbenicillin) Sigma cat # 1389DL-p-Chlorophenylalanine Sigma cat # C6506Media Recipes:YEG recombination pla ...

RNA interference (RNAi) is a biological process in which the introduction of double-stranded RNA (dsRNA) into a cell results in targeted post-transcriptional gene silencing.Historically RNAi has been ...

We use the Promega Ribomax Large Scale RNA Production System T7 (Cat. No.: P1300) to produce dsRNAs and the RNAi Exp.html" target=_blank Jack Dixon protocol ( RNAi _Dixon.html" target=_blankdownloaded ...

Chemicals needed:Glucose L-( )-Arabinose Sigma Cat # A3256 L-Rhamnose Sigma cat # R3875 Cb (carbenicillin) Sigma cat # 1389DL-p-Chlorophenylalanine Sigma cat # C6506Media Recipes:YEG recombination pl ...

The Easiest Route to Guaranteed SilencingIncreasing numbers of researchers are using small interfering RNAs (siRNAs) to reduce the expression of specific mammalian genes. Applications of siRNA induced ...

Restriction Map and Cloning Site of the RNAi-Ready pSIREN-DNR Vector. Unique restriction sites are in bold. RNAi-Ready pSIREN-DNR is provided as a linearized vector digested with BamH I and EcoR I. N ...

RNA Marker ( RL1000; RL6000; RL10000) 可以进行一般琼脂糖凝胶电泳和变性琼脂糖凝胶电泳。以RL10000为例，具体使用方法如下：普通琼脂糖凝胶电泳时1.按下列组份配置RNA Marker样品。2.均匀混合后65℃加热10分钟，迅速冷却至室温（最好用PCR仪)。3.使用高质量的琼脂糖，用1×TAE Buffer制备3%凝胶，制胶时凝胶中请加入溴乙锭(最终浓度：1 ...

在人细胞中快速、高水平地表达目的蛋白 在2至3个星期内即可获得高效价的病毒 无需噬斑纯化 靶细胞可以是分裂或不分裂的细胞（dividing/or nondividing cell） CLONTECH的Adeno-X™表达系统是一个特别 高效的腺病毒表达系统，用于在人细胞中瞬时及高水平地表达目的蛋白质。由于Adeno-X™病毒DNA中含有特别设计的克隆位点，你可以在2星期内获得高效价的腺病毒 ...

1．分别设立两个反应，用适当的限制性内切核酸酶消化1~10μg质粒DNA和外源DNA片段，使它们能产生平末端。2．苯酚：氯仿抽提与乙醇沉淀法纯化出已被消化的载体和外源DNA片段。3．分别用TE（pH 8.0）重新溶解纯化出的两种DNA沉淀，使终浓度为100 ng/ml。 假设1 bp相当于660 Da，计算DNA的终浓度（pmol/ml）。4．将载体DNA去磷酸化。5．按下表将适量的DNA转移至无 ...

体外定点突变技术是研究蛋白质结构和功能之间的复杂关系的有力工具，也是我们在实验室中改造/优化基因常用的手段。蛋白质的结构决定其功能，二者之间的关系是蛋白质组研究的重点之一。对某个已知基因的特定碱基进行定点改变、缺失或 者插入，可以改变对应的氨基酸序列和蛋白质结构，对突变基因的表达产物进行研究有助于我们了解蛋白质结构和功能的关系，探讨蛋白质的结构/结构域。而利用定点突变技术改造基因，相信 ...

TRTn3 terminal inverted repeatslacZ5'-truncated lacZ gene encoding beta-galactosidaseLEU2LEU2 gene from S. cerevisiaeampEncodes beta-lactamaseloxPlox site target for Cre recombinaseUses: Gene disrupti ...

TRTn3 terminal inverted repeatsHAHemagglutinin (HA) epitope loxRlox site target for Cre recombinaselacZ5'-truncated lacZ gene encoding beta-galactosidaseURA3URA3 gene from S. cerevisiaetetTetracycline ...

Identify appropriate celltype over-expressing corresponding gene.Find out if transcription can be stimulated further eg by induction (note: the higher the mRNA level of the gene in question the easier ...

Plasmid isolated by this procedure can be used routinely for electrophoretic analysis restriction endonuclease digestion and transformation of E. Coli. sequencing PCR and most other molecular biologic ...

The gel shift or electrophoretic mobility shift assay provides a simple and rapid method for detecting DNA-binding proteins. This method has been used widely in the study of sequence-specific DNA-bind ...

1) The ligation mixture contains the following: vector DNA (~100 ng) insert DNA (equimolar or 2 or 3 X molar concentration of vector) water added to 18 µl 2) Heat the mixture at 45 °C for 5 min. to me ...

Prepare a ligation mix:Ligation Mix (2x)10x ligase buffer1.0 mldigested vector(0.1 mg/ml)1.0 mlH2O6.0 mltotal8.0 mldivide ligation mix into two Eppendorf tubesLigation Rxn InsertControlligation mix ...