RNA实验技术

一.基本原理　　核酸分子杂交技术是目前分子生生物学、细胞生物学和生物化学研究中应用最广泛的技术之一，是定性、定量和定位检测两条来源不同的聚核苷酸链上碱基顺序同源性的一种手段。DNA分子是高度有序的双键分子。一条链的碱基与另一条链的碱基以氢键配对相连．形成腺嘌呤与胸腺嘧啶(A．T)，鸟嘌呤与胞嘧啶（G.C）的特定碱基对；在RNA中则为A与U(尿嘧啶)，G与C配对。　　在碱性环境加热或加入变性剂的条件 ...

OverviewThe PRINS (Primed in situ) technique uses a specific primer dNTPs with Dig-11-dUTP and DNA polymerase to perform a primer extension reaction on a chromosome preparation. To date it has been us ...

IntroductionPolytene chromosomes are present in many larval tissues in Drosophila. They result from subsequent rounds of DNA replication not followed by cell division and they represent a material of ...

IntroductionTechniques routinely used in our lab for analysing the X-inactivation process in differentiating ES cells are described. We focus particularly on chromatin changes (histone modifications p ...

I. DNA oligonucleotide probes: DNA oligonucleotides are the preferable probes at present. They are synthesized chemically which allows for the incorporation of amino-modified nucleotides (amino-allyl ...

IntroductionAn increasing body of evidence indicates that the spatial positioning of genes in the interphase nucleus is highly relevant for their function (Lanctot et al 2007; Meaburn & Misteli 2007; ...

IntroductionAn increasing body of evidence indicates that the spatial positioning of genes in the interphase nucleus is highly relevant for their function (Lanctot et al 2007; Meaburn & Misteli 2007; ...

I. General considerations: In situ hybridization of yeast cells is almost identical to mammalian cells except that the cell wall has to be removed by spheroplasting the cells prior to hybridization. T ...

I. IntroductionMost current methods of measuring gene expression rely on averaging many cellular responses or artificial amplification steps to reach a detectable threshold of signal. In contradistinc ...

OverviewThis protocol is a modification of the standard FISH protocol published by Hiro Hirai and Phil LoVerde in Parasitology Today (1995 11(8) p 310-314) that has been optimised for use with BAC pro ...

In contrast with other methods using gonads affixed to slides the various manipulations described below (gonad dissections hybridizations and washes) are all done in solution either in a glass dishes ...

I. General Considerations:-Decide which radiolabelled UTP you will use.32P is cheap available every week (HFI) and gives good signal rapidly but with only fair resolution at the tissue level. Losses a ...

These protocols describe non-radioactive methods for in situ hybridization on frozen sections whole mount embryos and on cultured cells. They have been freely adapted and modified to greater or lesser ...

This procedure was adapted from one originally described by Tautz and Pfeifle (1989). It makes use of digoxigenin labelled probes and alkaline phosphatase-conjugated anti-digoxigenin antibody fragment ...

Remove slides from freezer thaw for 5 min. at 55°C. Fix 10 min. in 4% paraformaldehyde 4°C. Wash 5 min. in 0.5x SSC RT. Immerse slides in proteinase K solution 1-5 µg/ml in RNase Buffer for 10 min. R ...

Pipet 12.5µl 35S-UTP (1200 Ci/mmol) into 1.5ml microfuge tube. Final concentration should be 12µM. Lyophilize in speed vac. (Do not use 35S-UTP that has been previously thawed. Order 250µCi vials and ...

Harvest tissue and rinse in PBS or saline. Immerse tissue in 4% paraformaldehyde/0.1M sodium phosphate buffer pH7.4 (recipe follows) at 4°C for 1-3hrs. Try to avoid overnight fixation if possible as t ...

1. Collect embryos dechorionate and fix in a (1:1) mixture of heptane:fix. Fix is 5% formaldehyde 0.05M EGTA in 1X PBS. pH 7.0. Devitellinize embryos by shaking in an equal mixture of methanol:heptane ...

PRE-TREATMENTFix the cells with Carnoy’s and drop them onto a glass slideAir dry (and store in -20 ºC until use)Age (if cells are fresh) the cells in 2xSSC 30 min 37 ºCTreat with 10-50 µg/mL pepsin in ...

a) 2-5 µg of plasmid DNA containing the cDNA insert is linearized using an appropriate restriction enzyme. For antisense probes a unique restriction site 5' to the insert is used. This digested DNA wi ...