/html/DNA/dnaothers/index.html DNA实验 / DNA其他技术 zh-cn <a href='http://www.5ibio.com' target='_blank'>Power by DedeCms</a> jurgenyan##yahoo.com.cn /html/DNA/dnaothers/20070731/12202.html 限制性内切酶能特异地结合于一段被称为限制性酶识别序列的DNA序列之内或其附近的特异位点上,并切割双链DNA。它可分为三类:Ⅰ类和Ⅲ类酶在同一蛋白质分子中兼有切割和修饰(甲基化)作用且依赖于ATP的存在。Ⅰ类酶结合于识别位点并随机的切割识别位点不远处的DNA，而Ⅲ类酶 2007-07-31 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070730/12197.html DNA分子水平上的多态性检测技术是进行基因组研究的基础。RFLP(Restriction Fragment Length Polymorphism,限制片段长度多态性)已被广泛用于基因组遗传图谱构建、基因定位以及生物进化和分类的研究。RFLP是根据不同品种（个体）基因组的限制性内切酶的酶切位点碱基发生突 2007-07-29 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070719/12168.html 大分子核酸的检测通常使用琼脂糖凝胶层析，但是由于其分辨率不够，所以检测小分子核酸使用聚丙烯酰胺凝胶（PAGE）层析，分辨率可以达到几个bp。对于PAGE胶的一些技术参数罗列如下表： 2007-07-19 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070719/12167.html 琼脂糖凝胶层析的技术数据 琼脂糖凝胶作为核酸检测主要也是理想材料，使用十分广泛，其理化性质一些参数如下表： 2007-07-19 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070528/11846.html 分清胶条的正负极，轻轻地将IPG胶条胶面朝下置于聚焦盘或水化盘中样品溶液上，使得胶条的正极（标有+）对应于聚焦盘的正极。确保胶条与电极紧密接触。不要使样品溶液弄到胶条背面的塑料支撑膜上，因为这些溶液不会被胶条吸收 2007-05-28 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070112/2602.html 是近年发展起来的研究核酸与蛋白质相互作用简单、快速、敏感的方法。目前已经成为转录因子研究的经典方法 2007-01-12 DNA其他技术 秩名 faculty.virginia.edu /html/DNA/dnaothers/20070112/2601.html BACDNApreparation MiniprepwithAutoGen740 Innoculate3-4mlLBwith20g/mlchloramphenicol Growat37°Covernightwithshaking IsolateBACDNAwithAutoGen740 ResuspendBACDNAin50lTEpH8.0or50lddH 2 O MiniprepwithQia 2007-01-12 DNA其他技术 秩名 www.tree.caltech.edu /html/DNA/dnaothers/20070112/2600.html DNA fingerprinting is sometimes called DNA typing. It is a method of identification that compares bits of DNA. A DNA fingerprint is constructed by first drawing out a DNA sample from body tissue or fluid such as hair, blood, or saliva. 2007-01-12 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070112/2599.html AFLP: not only for fingerprinting, but for positional cloning 2007-01-12 DNA其他技术 秩名 carnegiedpb.stanford.edu /html/DNA/dnaothers/20070112/2598.html There are multiple variations to this protocol, but we find that this one works well in all cases we tested. 2007-01-12 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070112/2597.html Design complementary oligonucleotides with compatible half sites on the ends (I use BamHI and BglII 2007-01-12 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070112/2596.html DNAse requires Mg, some factors are inactivatedby it! Remember ug/KB x 0.66 = picomole thus 1ng of 300bp probe =2 femptomole 2007-01-12 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070112/2595.html Probe preparation: see endlabl.ptc & isotach.ptc. probe shouldbe 10&copy;20k cpm/ul 2007-01-12 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070112/2594.html Electrophoretic Mobility Shift Assay (EMSA) 2007-01-12 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070109/2548.html Purification of oligonucleotides (which have already been purified by reverse phase) can increase the efficiency of site directed mutagenesis from around 30% to ~75% or greater in some cases 2007-01-09 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070109/2547.html 32P ATP标记寡核苷酸，Wear gloves throughout and work in radiation area. Monitor area before and after use. 2007-01-09 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070109/2546.html 寡核苷酸的纯化 2007-01-09 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070108/2529.html 凝胶迁移或电泳迁移率检测（Electrophoretic Mobility Shift Assay，EMSA）是一种检测蛋白质和DNA序列相互结合的技术，最初用于研究DNA结合蛋白和其相关的DNA结合序列相互作用，可用于定性和定量分析。 2007-01-08 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070108/2528.html Wash one 0.45um Millipore nitrocellulose filter (Cat# HAWP01300) in 1ml 0.4M KOH for 1 min; wash in 3ml dH2O for >1min 2007-01-08 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070108/2527.html 双链 DNA 能够通过硝化纤维膜 (NC)，但是 DNA-protein 复合物不能通过NC filter. 通过定量分析留在滤膜上的复合物，可以判断二者的结合常数 2007-01-08 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20070108/2526.html Combine an appropriate concentration of probe that has been labeled on one strand with the appropriate concentration of protein in a total volume of 50ul at 1x Binding Buffer conditions 2007-01-08 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1221.html 目前大多数研究者遵循一条规则，即10个单位的内切酶可以切割1μg不同来源和纯度的DNA。通常，一个50μl的反应体系中，1μl的酶在1X NEBuffer终浓度及相应温度条件下反应1小时即可降解1μg已纯化好的DNA。如果加入更多的酶，则可相应缩短反应时间；如果减少酶的用量，对 2006-10-18 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1220.html 体外定点突变技术是研究蛋白质结构和功能之间的复杂关系的有力工具，也是我们在实验室中改造/优化基因常用的手段。蛋白质的结构决定其功能，二者之间的关系是 蛋白质组 研究的重点之一。对某个已知基因的特定碱基进 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1219.html PreparationofNestedDeletions Procedure: 1)Needtocut10gofplasmidintwospots('A'and'B')inpolylinker.'A'cutis'itharestrictionenzymewhichgivesa3'recessedend(exonucleasesensitive)nexttoinsert.Since3'recess 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1218.html QuikChange(Stratagene)Thisisaquickandreliable(anddeadeasytoo!)methodforPCRbutcostsmore.Itis,however,highlyrecommendedifyouneedtomakeonlyasmallnumberofmutants,orifyoucan'torwon'tdoanymolecularbiologyth 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1217.html MutagenesisbyPCR(adaptedfromIto etal ,Gene102,67-70) Thismethodneedsonlyasinglemutagenicprimer(+3otherprimerswhicharethesameforallconstructsifusedinthesamevector)andwouldbeacheapandreliablemethodiflot 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1216.html EXO/S1DELETIONSERIES 1.CutDNAwith5'-and3'-overhangs,gelcheck,phenol-SevagextractandNaOAc/EtOHppt. 2.PrepareoneS1nucleasetube(onice)foreachtimepoint:15mlS1Buffer+0.25US1/ml(5U). 3.Use5ml(=0.5mg)digest 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1215.html DpnImediatedsite-directedMutagenesis AhighlyeffectivesimplemethodformakingsitedirectedlesionsinplasmidswithoutsubcloningbasedontheworkofFisherandPei(1997). 1.AmplificationofmutantDNA DNAtemplateplasm 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1214.html Erase-a-Base System TheErase-a-Base SystemisdesignedfortherapidconstructionofplasmidorM13subclonescontainingprogressiveunidirectionaldeletionsofanyinsertedDNA.Thesystemisbasedontheproceduredevelopedb 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1213.html CreatingadeletionbyPCRsplicing Contributedby Dr.A.Gratchev Ididn'twanttoplacethisinthemethodssectionduetoitssimplicity.TodeleteadesiredfragmentfromexistingDNAfragmentallyouneedisapairofprimerscombine 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1212.html Invitro site-specificmutagenesis MutagenesisisafundamentallyimportantDNAtechnologywhichseekstochangethebasesequenceofDNAandtestitseffectongeneorDNAfunction.Themutagenesiscanbeconducted invivo (instud 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1211.html 甲基化研究对于基因的 调控和肿瘤的发生有非常密切的关系。在一般正常的细胞中，基因调控区CpG岛处于非甲基化的状态。而当细胞发生癌变后，这些CG区域往往呈现甲基化状态。英国医学刊物《theLancet》报道奥地利因斯 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1210.html DNAmethylationisanepigeneticeventthataffectscellfunctionbyalteringgeneexpressionandreferstothecovalentadditionofamethylgroup,catalyzedbyDNAmethyltransferase(DNMT),tothe5-carbonofcytosineinaCpGdinucle 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1209.html MethylatedCpGIslandAmplification ProtocolwrittenbyMinoruToyota 2.Materials 2.1.MCA RestrictionenzymesSmaI,XmaI T4DNAligase TaqDNApolymerase 10XPCRreactionbuffer: 670mMTris-HCl,pH8.8 40mMMgCl 2 160mMN 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1208.html Methylation-SpecificPCR ProtocolwrittenbyJamesHerman* Methylation-specificPCR(MSP)isasimplerapidandinexpensivemethodtodeterminethemethylationstatusofCpGislands.Thisapproachallowsthedeterminationofmet 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1207.html BisulfiteTreatmentofDNA AdaptedfromFrommeret.al.* DiluteDNA(upto2mg)into50mlwithdistilledH 2 O. Add5.5mlof2MNaOH. Incubateat37°Cfor10minutes(tocreatesinglestrandedDNA). Add30mlof10mMhydroquinone(Sig 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1206.html DNA-MethyltransferaseAssay ThisprotocolwaswrittenbyJean-PierreIssa,basedonAdamsetal.* Kam-WingJairhasmadesomeusefulshortcutsthatworkwellifyouarecareful.Hereis Kam-WingJair'sversionoftheassay . Thisas 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1205.html BisulfiteModificationofDNA Source: ProtocolOnline Abstract: ModifyingDNAusingsodiumbisulfitetoconvertunmethylatedcytosinestouracilsandsubsequentlydetectmethylatedcytosinesusingmethylationspecificPCR( 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1204.html 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1203.html DNaseIFootprintingassayisamethodofstudyingDNA-proteininteractionandidentifyingtheDNAsequencetowhichaproteinbinds.First,atargetDNAfragmentabout100-300bpinlengthiseitherPCRgeneratedorcutfromavectorandt 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1202.html Author: Long-ChengLi Source: ProtocolOnline Abstract: SimplifiedmethodforpreparingG+Aladderrunalongwithfootprintingreaction.It'smuchsimplethantheoriginalMaxima-Gilbertsequencingreactionandworksfine. 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1201.html DNaseIFootprintint Solutions 10XBindingBuffer 200mMTris8.0200 m l1MTrispH8.0 500mMNaCl100 m l5MNaCl 10mMEDTA20 m l0.5MEDTApH8.0 680 m lQ storeatroomtemperature DNaseIDilutionBuffer (thisbufferprovide 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1200.html Footprinting FootprintingisamethodfordeterminingtheexactDNAsequencetowhichaparticularDNA-bindingproteinbinds.Examples: hormone-receptorcomplexesthatbindtotheir hormoneresponseelements transcriptionfa 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1199.html Overview AnendlabeledDNAprobeisincubatedwithapurifiedDNA-bindingfactororwithaproteinextract.TheunprotectedDNAisthendigestedwithDNaseIsuchthatonaverage,everyDNAmoleculeiscutonce.Digestionproductsareth 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1198.html EMSAusingdsOligonucleotides Solutions 10XAnnealingBuffer 200mMTris8.0200 m l1MTrispH8.0 10mMEDTA8.020 m l.5MEDTApH8.0 500mMNaCl100 m l5MNaCl 280 m lQ storeatroomtemperature 10XKlenowBuffer 500mMTris7 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1197.html GelShiftAssaySystems Thegelshift,orelectrophoreticmobilityshift,assayprovidesasimpleandrapidmethodfordetectingDNA-bindingproteins.Thismethodhasbeenusedwidelyinthestudyofsequence-specificDNA-bindingpr 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1196.html BAND-SHIFT Theproceduredescribedinthischapterforthedeterminationofaffinityconstantsandkineticdissociationconstantsbyband-shiftassayreferstoanidealantibodyfragment(e.g.,ascFvoranFabfragment)bindingtoa 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1195.html 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1194.html 2006-09-27 DNA其他技术 秩名 生物实验网 /html/DNA/dnaothers/20061111/1193.html RFLP操作要点 一、材料 基因组 DNA(大于50kb,分别来自不同的材料)。 二、设备 电泳仪及电泳槽，照相用塑料盆5只，玻璃或塑料板(比胶块略大)4块，吸水纸若干，尼龙膜(依胶大小而定)，滤纸，eppendorf管(0.5ml)若干。 2006-09-27 DNA其他技术 秩名 生物实验网