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Polymerase III in vitro Transcription Steve Hahn For the following reactions use appropriate shielding and dispose of radioactive waste properly! A 20 microliter transcription reaction contains: 4.0 ...

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 ...

pSico Oligo Design Click on the Mac OSX program pSicoOligomaker 1.5 to select and design oligos for pSico and plentilox3.7. To operate it simply paste your sequence in the "sequence" window ...

Run-on transcription monitors the regulation of transcription in isolated nuclei. A. Preparation of Nuclei - (do everything at 0℃ to 4℃ in 50 ml tubes) 1. Pellet between 30 and 300 million cells at 15 ...

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 ...

Add 0.1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at 37℃. Autoclave for 15 minutes to remove any trac ...

3' R apid A mplification of c DNA E nds (RACE ) PCR This technique is used to obtain the 3'end of a cDNA it requires some sequence information internal to the mRNA under study. The sequence inf ...

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 ...

Steve Hahn. last modified Sat Oct 17 1998 Mix the following in an 0.5 ml eppendorf tube: 10-20 micrograms total yeast RNA 10 microliters hybridization mix H2O to bring the final volume to 25 microlite ...

1 ml linearized DNA template pBS-SK- ERD (X-E) (0.1-0.5 mg) 7.8 ml DDW 5 ml 5X transcription buffer ( stratagene ) 1 ml 750 mM DTT 1 ml Rnasin (RNase inhibitor; 15U/ml) 1 ml 10 mM ATP (pH 7) 1 ml 10 m ...

Ambion and Applied Biosystems have joined forces to provide a complete convenient solution for performing gene silencing experiments and validating the results by real-time RT-PCR. Ambion's Silencer&t ...

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 ...

RNase Protection Assay (RPA) BD Biosciences公司， PDF文件 http://www.bdbiosciences.ca/canada/downloads/protocols/RPA.pdf ...

The use of small interfering RNAs (siRNAs) to induce targeted gene silencing in mammalian cells offers researchers a powerful tool for analyzing gene function. Ideally one would like to work with indi ...

TRIzol method for RNA isolation -To tissue or cells add 200- 1 ml TRIzol (GIBCO BRL) 1.homogenize for 60 sec (vortex or polytron) 2.add 40ul-200 ml chloroform (1/5 volume of Trizol) 3.mix by vortexing ...

RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA (Figure 1 ...

检测基因特异阻断的最常用的方法是进行westernblot分析，比较导入siRNA 前后蛋白表达水平的变化。在一些情况下可能使用检测报告子基因的报告子系统例如β-半乳糖苷酶。也可以应用实时PCR（例如通过使用LUX™引物）或者其它类型基于细胞的分析检测细胞中转录本的水平。 ...

S1 Nuclease Protection Assay End-label oligo (20-25 mer) 4 µl 5X T4 Kinase Buffer 5 µl ATP (7000 Ci/mmol) 10 µl water + oligo (200 ng) 1 µl T4 Kinase 1. 37 deg C for 1 hour. ...

Steve Hahn Last Modified Fri Apr 25 2003 Wear gloves throughout use RNAse free solutions (either autoclaved or sterile filtered) and clean bench and pipetmen with 95% ethanol before use to eliminate ...

Gregory Hannon Cold spring harbor lab Our overall approach is to use an RNA polymerase III promoter to drive expression of encoded short hairpin RNA (shRNA). For this purpose we use the human U6 snRNA ...