MakingRNAprobesforinsituhybridization
Make DNA templates via PCRDay 1It's preferable to start with constructs that contain RNA polymerase start sites (T3, T7, or SP6) which allow use of primers outside the start-point. This gives the enzyme some docking space, improving transcription efficiency. If this is not possible, use extended primers with RNA-polymerase start sites in their 5' tails.Purify PCR products with QiaQuick PCR purification kit an......閱讀全文
Making－RNA－probes－for－in－situ－hybridization
Make DNA templates via PCRDay 1It's preferable to start with constructs that contain RNA polymerase start sites (T3, T7, or SP6) which allow use o
In－situ－hybridization
Note:?Although it is possible to perform in situ hybridization on bleached embryos, it appears to reduced the strength of the signal. For best results
Basic－Fluorescent－in－situ－Hybridization－(FISH)
實驗概要Fluorescence ?in situ hybridization method is a kind of physical map drawing method, ?use fluorescent element mark probe, to detect probe and spli
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
Fluorescence－In－Situ－Hybridization－using－TSA?
實驗概要This ?protocol describes steps for fluorescent in situ hybridization (FISH) ?to Drosophila embryos using Tyramide Signal Amplification (TSA?), and
細胞遺傳學——原位雜交(ISH)
In Situ Hybridization· ????????In Situ Hybridization?(jsmith1@po-box.mcgill.ca)In situ?hybridization, as the name suggests, is a method of localizing,
DNA微序列技術
·?????????Protocols for Making Drosophila Arrays?(Stanford U.)Detailed protocol for making arrays including PCR Amplification of cDNAs for Printing,?
熒光原位雜交（Fluorescence－in－situ－hybridization,FISH）
實驗原理熒光原位雜交（Fluorescence in situ hybridization FISH）是一門新興的分子細胞遺傳學技術，是20世紀80年代末期在原有的放射性原位雜交技術的基礎上發展起來的一種非放射性原位雜交技術。目前這項技術已經廣泛應用于動植物基因組結構研究、染色體精細結構變異分析、病
熒光原位雜交（Fluorescence－in－situ－hybridization,FISH）原理
2）標本變性①將制備好的染色體玻片標本于 50oC培養箱中烤片2～3h。（經Giemsa染色的標本需預先在固定液中退色后再烤片）。②取出玻片標本，將其浸在70～75oC的體積分數70％甲酰胺/2×SSC的變性液中變性2～3min。③立即按順序將標本經體積分數70％、體積分數90％和體積分數100％冰
熒光原位雜交（Fluorescence－in－situ－hybridization,FISH）（圖）
實驗原理熒光原位雜交（Fluorescence in situ hybridization FISH）是一門新興的分子細胞遺傳學技術，是20世紀80年代末期在原有的放射性原位雜交技術的基礎上發展起來的一種非放射性原位雜交技術。目前這項技術已經廣泛應用于動植物基因組結構研究、染色體精細結構變異分析、病
雙重和多重原位雜交(hybridization－in－situ)技術
為了在同一標本上或同一細胞內同時檢測是否存在兩種或兩種以上的靶核酸序列。可應用雙重或多重原位雜交技術．即以兩種或多種標記探針與靶核酸雜交。然后利用不同的檢測手段分別顯示各種靶核酸的存在和分布。該技術與免疫組織化學技術中的雙重或多重標記相似，除了探針本身的特異性外，對結果的干擾主要來自標記物及檢測試劑
cDNA
·?????????cDNA Synthesis?(Crawford Lab)mRNA can be converted into DNA (copy DNA, cDNA) by annealing oligo-dT to the 3' poly-A tail that occurs on
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
原位雜交(In－Situ－Hybridization,ISH)與熒光原位雜交（二）
5．洗膜 取出塑料袋，用剪刀剪開，小心取出濾膜，立即浸入盛有2×SSC和 0.5%SDS溶液的盤中，室溫下漂洗5min。再將濾膜移入2×SSC和0.1%SDS溶液中，室溫下洗滌15min（輕輕搖動）。然后將濾膜移入 0.1×SSC和0.5%SDS溶液中；68℃輕輕搖動保溫2h，更換緩沖液后繼
原位雜交(In－Situ－Hybridization,ISH)與熒光原位雜交（五）
⑦60伏電泳過夜。 ⑧取出凝膠，水中浸泡2次，每次5min。 ⑨室溫下將膠浸到50mmol/L NaOH和10mmol/l NaCl中45min，水解高分子RNA，以增強轉印。 ⑩室溫下將膠浸到0.1mol/L Tris·HCl (Ph7.5)中45min，使膠中和。
原位雜交(In－Situ－Hybridization,ISH)與熒光原位雜交（三）
（1）DAN斑點雜交①先將膜在水中浸濕，再放到15×SSC中。②將DNA樣品溶于水或TE，煮沸5min，冰中速冷。③用鉛筆在濾膜上標好位置，將DNA點樣于膜上。每個樣品一般點50μl（2～10μg DNA）。④將膜烘干，密封保存備用。（2）RNA斑點雜交：與上法類似，每個樣品至多加10μg總RNA（
原位雜交(In－Situ－Hybridization,ISH)與熒光原位雜交（六）
夾心雜交法可用濾膜和小珠固定吸附探針，使用小珠可更好地進行標準化試驗和更容易對小量樣品進行操作。Dahlen 等利用微孔板進行夾心雜交，可同時進行大量樣品檢測，他們先吸取DNA探針加到凹板中，然后用紫外線照射使其固定到塑料板上。用微孔板進行夾心雜交還可直接用于PCR技術。應用光敏生物標記探針
原位雜交(In－Situ－Hybridization,ISH)與熒光原位雜交（四）
（2）硝酸纖維素濾膜吸印。①將膠切成合適大小，切去右上角作為記號。②將膠放進盛有變性緩沖液（1.5mol/l NaCl, 0.5mol/L NaOH）的盤中輕搖動15min。③換到中和緩沖液（1mol/L Tris·HCl , pH8.0, 1.5mol/L NaCl）中輕搖動30min。④裁一張硝
原位雜交(In－Situ－Hybridization,ISH)與熒光原位雜交（一）
是用標記的核酸探針，使用非放射檢測系統或放射自顯影系統，在組織切片、細胞涂片及染色體制片上等對核酸進行定性、定位和相對定量研究的一種分子生物學方法，具有靈敏、特異、直觀等優點。已逐漸成為分子生物學和分子病理學的常見技術之一，廣泛應用于腫瘤生物學、血液病理學、遺傳、微生物學、細胞和分子生物學、神經內分
Southen雜交
Southern雜交One important thing for transfer:the weight of the object resting on top of the blotting apparatus should not exceed the weight equal to a 5
Preparation－of－nucleic－acid－probes
Preparation of nucleic acid probesIn standard nucleic acid hybridization assays the probe is labeled in some way. Nucleic acid probes may be made as s
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
Making－Metaphase－Spread
Materials:Colcemid (GIBCO: cat #: 15210-040)Hypotonic solution (pre-warmed 37C):? 1:1???? 0.4% KCl? +? 0.4% Sodium citrateFixative:? 3:1?? MeOH + Acet
Overview－of－telomerase－RNA－component－gene－hTerc－Transcriptional－Regulation
Telomerase is an enzyme which replicates the terminal sequences of eukaryotic chromosomes, namely the telomeres. Cells which have an unlimited replica
原位雜交組織化學實驗技術6
二、快速原位雜交細胞化學技術 原位雜交免疫細胞化學技術存在的難點之一是實驗手續繁瑣，實驗周期長。國外Liesi等（1986）和國內學者何彬等應用光敏生物素-鏈親合素（Biotin-streptavidin）膠體金系統進行原位雜交，得到了快速滿意的結果，全部實驗可在數小時內完成。 作用把含某種多肽
FISH－protocols－for－Drosophila2
?3.?Methods3.1 RNA Probe Preparation1.?? Different strategies can be used to prepare template DNA for synthesizing antisense RNA probes by?in vitro?tr
COLONY－HYBRIDIZATION
COLONY HYBRIDIZATION1) CUT 4 PIECES OF 3MM WHATMAN PAPER AND PLACE EACH ONE IN A SEPARATE CONTAINER(A CAFETERIA TRAY WILL PROBABLY WORK PERFECTLY).2)
Colony－Hybridization
ProcedurePrepare serial 10-fold dilutions of transformed bacteria in LB and spread 100 μL onto LB/Amp plates, as described in the Electroporation prot
DNA轉化
DNA轉化Chemical Transformation·?????????Transformation of Competent Cells (RbCl2 Method)?(Goldberg Lab)Very nice protocol for E. Coli transformation inc
Principles－of－nucleic－acid－hybridization
Principles of nucleic acid hybridization5.2.1.?Nucleic acid hybridization is a method for identifying closely related nucleic acid molecules within tw