RNAFISHonculturedcellsininterphase2
Post-labeling DNA processing and purificationQiagen PCR clean up to get rid of unused oligonucleotides;Add 20µl cot1DNA, 10µl ssDNA to compete for repetitive elements;Precipitate the probe with 1/10 V 3 M NaOAc pH5.2, 2.5 V 100% ethanol at -80°C for 30 minutes;Spin down pellet at 4°C for 20 minutes;Wash pellet with 70% ethanol;Resuspend pellet in 80µl hybridization mix.The probe is now ready to be used. It can be sto......阅读全文
RNA－FISH－on－cultured－cells－in－interphase
IntroductionFluorescence in situ hybridization (FISH) has become a widely used method in genome and molecular genetic studies. The technique is highly
RNA－FISH－on－cultured－cells－in－interphase2
Post-labeling DNA processing and purificationQiagen PCR clean up to get rid of unused oligonucleotides;Add 20µl cot1DNA, 10µl ssDNA to compete for rep
Fixation－of－Cells－Cultured－in－Transwell－Dishes
Fixation of Cells Cultured in Transwell DishesTranswell culture dishes are commonly used to culture cells so that the top and bottom of the cells can
HThymidine－Uptake－by－Cultured－Cells
H-Thymidine Uptake by Cultured CellsLEVEL IIMaterialsFibroblast cells in log phase growthCa, Mg free-phosphate buffered saline (PBSA)5% (w/v) Glutaral
3HThymidine－Uptake－by－Cultured－Cells
Materials Fibroblast cells in log phase growthCa , Mg free-phosphate buffered saline (PBSA)5% (w/v) Glutaraldehyde (GTA)2% (w/v) Perchloric Acid (PCA)
3HThymidine－Uptake－by－Cultured－Cells
3H-Thymidine Uptake by Cultured Cells OverviewA radioactive method for measuring cultured cell growth with 3H-thymidine is described here. MaterialFi
Multicolour－3DFISH－in－vertebrate－cells1
IntroductionMulticolour 3D-FISH in combination with confocal microscopy, 3D image reconstruction and quantitative image analysis is an efficient tool
Multicolour－3DFISH－in－vertebrate－cells5
Author NotesAfter the fourth round of DOP amplification the probe quality is considerably reduced.Use the low stringency cycles only in case you start
Multicolour－3DFISH－in－vertebrate－cells2
Small DNA-probes from cosmids or plasmids clonesThese kind of probes, especially plasmids, have become out of fashion for 3D-FISH due to their delicat
Multicolour－3DFISH－in－vertebrate－cells4
DetectionThe choice of the detection scheme depends on several factors: (i) the number of haptens and fluorochromes used for probe labeling; (ii) the
Multicolour－3DFISH－in－vertebrate－cells6
Back to topReviewer CommentsReviewed by: Luis Antonio Parada, CIC Biogune, Derio, Spain.In my experience the primers are better preserved when kept at
Multicolour－3DFISH－in－vertebrate－cells3
Pepsin treatmentEquilibrate slides (kept in 50%FA/2xSSC) in 2xSSC 2 minutes;Switch to 1xPBS 3 minutes;Pepsin: 3-5 minutes in 0.01 N HCl/0.002% pepsin.
细胞遗传学——原位杂交(ISH)
In Situ Hybridization· In Situ Hybridization (jsmith1@po-box.mcgill.ca)In situ hybridization, as the name suggests, is a method of localizing,
Isolation－of－microRNA－(miRNA)
实验概要 This protocol utilizes the powerful guanidine isothiocyanate–phenol:chloroform extraction method which allows the rapid isolation of t
Global－Expression－Profiling－of－RNA－from－Laser－Microdissected－Cells－at－...
Global expression profiling of RNA isolated from laser microdissected cells allows one to profile a specific set of cells allowing for enhanced se
用细胞爬进行RNA－FISH检测的实验操作
用细胞爬片进行 RNA FISH 检测的实验流程 1. 细胞在盖玻片上进行培养，细胞长好后用 CSK 缓冲液简单洗涤。 2. 细胞爬片用 4% 多聚甲醛在 4℃ 固定 10 min。 3. 用含有 0.5% TritonX-100,，10 mM VRC 的 CSKBuffer 溶液在 4℃ 处理 1
以－RNA－为靶目标－FISH－探针的标记实验
标记的 RNA、DNA 或者核酸类似物可以通过荧光原位杂交（FISH) 的方法检测细胞标本内的 RNA。目标 RNA 通常是由基因组 DNA 转录来的信使 RNA(mRNA)。由于髙特异的核酸碱基互补配对原则，特定的 RNA 分子可以从众多共表达的 mRNA 分子中筛选出来。对选择的 mRN
以－RNA－为靶目标－FISH－探针的标记实验
标记的 RNA、DNA 或者核酸类似物可以通过荧光原位杂交（FISH) 的方法检测细胞标本内的 RNA。目标 RNA 通常是由基因组 DNA 转录来的信使 RNA(mRNA)。由于髙特异的核酸碱基互补配对原则，特定的 RNA 分子可以从众多共表达的 mRNA 分子中筛选出来。对选择的 mRNA 分子
TRIZOL提取注意事项3
3.台式离心机最大能达到2,600×g的离心力，如果将离心时间延长到30-60分钟可以满足步骤2和步骤3中的操作。疑难解答：RNA抽提每1 mg组织或1×106培养细胞预期的RNA产量1.肝和脾，6—10μg2.肾，3—4μg3.骨骼肌和脑组织，1—1.5μg4.胎盘，1-4μg5.上皮细胞(1×1
In－Situ－Hybridization－to－Somatic－Chromosomes－in－Drosophila
In Situ Hybridization to Somatic Chromosomes in DrosophilaAbby F. DernburgINTRODUCTIONIn situ hybridization was originally developed as a technique fo
PriCells:－Questions
Questions 1: Isolation of hepatocytes from other speciesThe basic two-step perfusion procedure can be used for obtaining hepatocytes from various r
Isolation－of－Total－RNA－from－Animal－Cells－use－RNeasy－Mini－Kit
实验概要Extract the total RNA from animal cells by using RNeasy Mini Kit (QIAGEN No.74104) 主要试剂SDS based extraction solution实验步骤1. Harvest cells.1) Try
一种新方法评定在培养细胞中siRNA效率
A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells.Wu W, Hodges E, Redelius J, Hoog C.Nucleic Acids Res. 200
一种新方法评定在培养细胞中siRNA效率
A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells.Wu W, Hodges E, Redelius J, Hoog C.Nucleic Acids Res. 200
E.Z.N.A.®－Total－RNA－Midi－Kit－Protocol－for－Eukaryotic－Cells－and－Tissues
实验概要E.Z.N.A.® Total RNA Midiprep Kit provides a rapid and easy method for the isolation of up to 600 ug of total RNA from cultured eukaryotic cells,
MicroRNA－Expression－Profiling－by－Bead－Array－2
Materials and MethodsCell Culture, Interferon Treatment, and RNA PrecipitationMelanoma cells (ME-15) were cultured in RPMI 1640 with L-Glutamine suppl
Differentiate－ES－cells－into－glial－cells－and－neurons
Day -1: Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.___________________Day 1: Trypsiniz
Culture－of－BEND－Cells－(Bovine－Endometrial－Cells)
Culture of BEND Cells (Bovine Endometrial Cells)Charles E. Krininger, III and Peter J. Hansen Dept. of Animal Sciences, University of FloridaThis prot
RNA－extraction－using－trizol/tri
RNA extraction with TRIzol (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) is a common method of total RNA extraction fro
Cell－Cycles－－Introduction
The onion root tip and the whitefish blastula remain as the standard introduction to the study of mitosis. The onion has easily observable chromosomes