DNA重组

六、核酸分子杂交实验因素的优化　　（一）探针的选择　　根据不同的杂交实验要求，应选择不同的核酸探针。在大多数情况下，可以选择克隆的DNA或cDNA双链探针。但是在有些情况下，必须选用其它类型的探针如寡核苷酸探针和RNA探针。例如，在检测靶序列上的单个碱基改变时应选用寡核苷酸探针，在检测单链靶序列时应选用与其互补的DNA单链探针（通过克隆人M13噬菌体DNA获得）或RNA探针，寡核苷酸探针也可。长的 ...

Transient Transfection of Cos-1 CellsNote: This protocol has been optimized for Cos-1 cells. Successful transfection of each cell type requires optimization of the basic protocol. Variables to consid ...

Chemical transformationPreparation of chemically competent cellsHave the following solutions at 0-4 deg C:a) 100 mM MgCl2b) 100 mM CaCl2-15% glycerolc) sterile GSA bottles and pre-cooled rotor1. Grow ...

Long Term Storage of Transformed E.coliTransfer 10 ml of 250 ml overnight culture in sterile flip-cap 15 ml Tube. Add sterile Glycerol to 15 % final conc. (i.e. 1.875 ml of 80% Glycerine stock solutio ...

The magic E. coli transformation protocolTake the ultra-competent cells out of freezer. Thaw on ice. Put your selection plates (LB +?) on the bottom shelf of an incubator to pre-warm them (about 37 to ...

pGL3 Luciferase Reporter VectorsThe pGL3 Luciferase Reporter Vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be ...

pGL3 Luciferase Reporter VectorsThe pGL3 Luciferase Reporter Vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be ...

TRANSFORMATION (ONE-STEP PEG METHOD)Dilute 1:100 a fresh O/N culture of bacteria into prewarmed LB broth and incubate cells with shaking (225 rpm) to an OD600 of 0.3-0.4. Add an equal volume of ice-co ...

Transformation of Competent CellsA. Preparing competent cells.Inoculate 30 ml of SOB broth in a 250-ml flask with bacteria to be transformed from a single colony on a fresh plate. Incubate overnight a ...

Transformation of E. coliReference: H. Inoue H. Nojima & H. Okayama Gene 96 23-28 (1990) 1. Culture E. coli in 250 ml of SOB medium in a 2-liter flask at 18 C with vigorous shaking until an A260 of 0. ...

Bacterial Colony PCRObjective:This protocol allows rapid detection of transformation success when primers are available to allow determination of correct ligation products by size or hybridization. Pr ...

Screening of recombinants It is best to do a test ligation without insert if the background is low it would not be necessary to screen colonies for insert.1) colony PCR1) Pick a colony from plate and ...

Stable Transfection (Electroporation)Outline:Electroporation can be used for both transient and stable transfection of mammalian cells. Cells are placed in suspension in an appropriate electroporation ...

基因组DNA Southern杂交1）基因组DNA Southern印迹的制备预备1．用适当的限制性内切酶消化基因组DNA样品（10μg）。2．进行琼脂糖凝胶电泳。一般用0.7～1.0％的琼脂糖凝胶分离基因组DNA，它在1～15kb的范围内有较好的分辨率，可选用TBE或TAE缓冲液。琼脂糖凝胶电泳需在1V/cm的电压下进行，如果要分离片段大小相似的DNA带，应用较大的凝胶（20×25cm）。常用的 ...

Restriction Map and Multiple Cloning Site of pEGFP-N2. (Unique restriction sites are in color or bold.) The Not I site follows the EGFP stop codon. The Nhe I site cannot be used for fusions since it c ...

Map of pLP-EGFP-C1 Vector. Unique restriction sites are shown in bold. ...

Restriction Map and Multiple Cloning Site of pbgal-Control. Unique restriction sites are in bold. ...

Restriction Map and Multiple Cloning Site of pbgal-Basic. Unique restriction sites are in bold. ...

Isolation of DNA from Acrylamide Gels1. Pour a vertical acrylamide gel using TEA buffer. A 4 % non denaturing gel is correct for most applications. 2. Run out DNA fragments. For fragments greater than ...

Oligonucleotide PurificationSteve HahnLast modified 10/16/98Purpose: Purification of oligonucleotides (which have already been purified by reverse phase) can increase the efficiency of site directed m ...