[生物工程精品] 粗毛栓菌漆酶cdna的pcr擴(kuò)增 論文

本科生畢業(yè)設(shè)計(jì)（論文）題目粗毛栓菌漆酶CDNA的PCR擴(kuò)增姓名學(xué)號(hào)系別生命科學(xué)系專業(yè)生物科學(xué)指導(dǎo)教師職稱教授年月日教務(wù)處制A1A0A2A3A4A5A6A8A9A7A11A10A12A13A14A15A16A17A18A19A20A21A22A23A24A25A26A27A28A29A30A31A32A33A34A35A36A37A38A39A40A41A42A43A44A45A46A47A48A23A49A50A27A51A52A49A53A54A55A56A57A58A55A56A59A60A61A27A62A63A26A64A65A27A66A67A68A40A35A36A27A69A40A70A71A38A39A40A43A72A73A74A75A76A77A40A70A71A38A39A40A78A79A80A33A34A35A36A81A82A83A41A42A24A84A43A85A86A87A86A87A86A87A88A88A89A90A91A26A27A31A32A33A34A35A36A27A40A70A71A38A39A40A41A42ABSTRACTTOTALRNAWASEXTRACTEDFROMTRAMETESGALLICACULTIVATEDFOR10DAYSINHIGHCLOWNMEDIUMTHERESULTSHOWEDTHATTWOCDNAFRAGMENTSWASAMPLIFIEDBYRTPCRKEYWORDSTRAMETESGALLICAFR,LACCASECDNA,RTPCRA92A93A94A95A96A97A98A99A100A101A102A103A104A105A106A107A108A109A110A111A112A113A114A115A116A117A118A119A120A121A122A123A124A125A126A127A128A120A129A130A131A132A133A134A135A136A137A138A131A139A140A120A141A142A143A144A137A145A146A147A148A149A150A151A152A153A121A122A154A155A120A156A157A137A158A131A151A159A160A145A146A161A162A149A150A151A155A163A120A164A165A137A153A161A111A151A166A167A168A169A170A110A118A164A165A137A162A171A162A172A173A118A174A175A170A103A104A105A106A176A177A109A178A179A180A173A118A181A182A109A183A184A185A186A187A176A188A189A109A178A179A180A183A190A191A192A193A109A183A194A188A109A178A179A180A195A196A197A198A199A200A201A202A203A204A205A203A206A207A208A209A210A211A204A212A213A210A214A215A216A217A218A216A219A220A221A222A223A224A222A225A226A217A227A228A229A230A231A232A233A234A235A236A237A238A239A231A236A239A240A241A242A243A244A245A246A236A239A247A234A235A222A248A249A250A251A222A252A253A254A255A235A222A66A0A1A2A235A222A9A3A4A3A208A5A6A240A7A66A8A217A75A10A11A244A231A12A13A14A15A16A17A18A19A20A21A22目錄摘要1關(guān)鍵詞1ABSTRACT1KEYWORDS1引言11材料與方法211主要儀器與試劑用品2111主要儀器2112主要試劑2113主要用品212菌株及培養(yǎng)方式2121菌株2122培養(yǎng)基313PCR引物314總RNA的提取與檢測3141總RNA的提取3142總RNA的甲醛變性瓊脂糖凝膠電泳4143總RNA純度與濃度的檢測415RTPCR4151逆轉(zhuǎn)錄4152總CDNA的PCR擴(kuò)增4153PCR擴(kuò)增產(chǎn)物的瓊脂糖凝膠電泳52結(jié)果與討論521總RNA電泳結(jié)果522總RNA純度與濃度523PCR擴(kuò)增結(jié)果6參考文獻(xiàn)6致謝7A23A24A25A26A27A28A29A30A31A32A33粗毛栓菌漆酶CDNA的PCR擴(kuò)增生物科學(xué)專業(yè)學(xué)生于林田指導(dǎo)教師孫迅摘要本實(shí)驗(yàn)以粗毛栓菌作為出發(fā)菌，進(jìn)行了漆酶CDNA的PCR擴(kuò)增。以半纖G13512G13044為G11911G9316，用G20652G11911G1314G8706培養(yǎng)基G15培養(yǎng)1G19D，G6922G19610菌G1009G1319，提取總RNA，G1582RTPCR。結(jié)果G7186G12046G17902G17819RTPCRG7389G1016G7477CDNAG10267G8585G15999擴(kuò)增出G7481。關(guān)鍵詞粗毛栓菌，漆酶CDNA，RTPCR擴(kuò)增PCRAMPLIFICATIONOFLACCASECDNAINTRAMETESECGALLICASTUDENTMAJORINGINBIOLOGYYULINTIANTUTORSUNXUNABSTRACTTOTALRNAWASEXTRACTEDFROMTRAMETESGALLICACULTIVATEDFOR10DAYSINHIGHCLOWNMEDIUMTHERESULTSHOWEDTHATTWOCDNAFRAGMENTSWASAMPLIFIEDBYRTPCRKEYWORDSTRAMETESGALLICAFR,LACCASECDNA,RTPCR引言漆酶G708LACCASE，LACG709G7171G980G12193G3822G18222G8699G2282酶G15G3324結(jié)G7512G2656G2163G14033G990與G7905物G6251G3363G15892G18252G8699G2282酶G451G2766G1095G2172物G15892G8986G19120G15025G15519G11345G4396G3324G11540G16780G3822G11468G1296G1055G3800A34A35A36。G4439G7171G7380G12628G2345的G3822G19120G8699G2282酶G15G980G14336G2559G73894G1022G19120G2419G4388G15G2010G5079于3G1022G20652度G1457G4444的G993G2528結(jié)G2524G1313G9869G15G8611G1022G19120G2419G4388G3324G1664G2282G7438G2058G1025G18129G7389G5468G18337要的作用。漆酶G14033G1664G2282G50A37G17902G178194G1022電G4388G17836G2419G6116G8712G15G5194G1000G1288G19555G11540G980G1135G18222G12879G5225物的G8699G2282A34A37A36。G4439G7380G7101G7171G1186漆G7653的G2010G8864物G1025發(fā)G10628的G15G19555G2530G1166G1216發(fā)G10628G980G1135G20652G12573G11507菌G1075G14033G2010G8864G17837G12193酶。G10628G3324G1166G1216G11705G17959G15漆酶G5203G8879G4396G3324于G6297G4388菌G451半G11705菌G2656G4388G3230菌G1025G15G1306G1866G1025G7380主要的生產(chǎn)G13785G7171G6297G4388菌G1025的G11345G14116菌A34A38A36，粗毛栓菌G708TRAMETESGALLICAG709G7171G980G12193G11345G14116G6297G4388菌，G3324G6117G3281G2010G5079G5203G8879，G7171G7484G7420的G980G12193主要生物G19489G16311G13785。G5062G7389研究表明，該菌產(chǎn)漆酶G14033力較G20652A34A39A36。漆酶G7101期的研究主要G19610G1025G3324G1319內(nèi)G2163G14033的研究，G19555G115402G19世紀(jì)7G19年代前G2530聚丙烯酰胺凝膠電泳G2656G2010G4388克隆G12573生物學(xué)技術(shù)的問世G15漆酶的研究G1075揭開了新的G980頁。G3324G3281外G5468G3822菌G12193的漆酶基因得到了克隆G5194G1000進(jìn)行了G2010析G2656鑒定G15G980G1135漆酶基因G1075實(shí)G10628了G1866異G9316表達(dá)A34A40A36。漆酶G3324G7420質(zhì)G13044G19489G16311G451微生物菌G1319形態(tài)形G6116G12573方面具G7389G18337要G2163G14033A34A40A36，普G17902微生物產(chǎn)漆酶的量及純度G993足以滿足大規(guī)模的工業(yè)應(yīng)用。要工業(yè)G2282生產(chǎn)漆酶，可G17902G17819把可G20652效產(chǎn)漆酶的基因與載G1319G18337組，然G2530導(dǎo)入受G1319細(xì)胞，形G6116穩(wěn)定轉(zhuǎn)G2282G4388，大批量培養(yǎng)，使漆酶進(jìn)行異G9316表達(dá)。因此必須進(jìn)行漆酶基因的測序，了G16311基因組G6116及酶切G1313G9869G12573，以便DNAG18337A41A42A43A44A45A46A47A48A49A50A51組時(shí)選擇G2524適的限G2058性內(nèi)切酶進(jìn)行G7389效切割。所以漆酶的模板CDNA提取及大量擴(kuò)增G6116為首要工作。傳統(tǒng)的模板DNA提取方法G7171采用去垢劑如SDSG12573G7481破G3363細(xì)胞組G2010，溶G16311細(xì)胞膜，使G15519G11345質(zhì)變性，再用G15519G11345酶KG7481消G2282去除G15519G11345質(zhì)，尤G1866G7171與DNA結(jié)G2524的G15519G11345質(zhì)，再用G18222氯仿抽提，然G2530用乙醇沉淀核G18252，供PCR實(shí)驗(yàn)用A52A53A54。本實(shí)驗(yàn)主要G7171擴(kuò)增G7389效表達(dá)漆酶的CDNAG10267G8585，因此利用逆轉(zhuǎn)錄的方法G2058備了模板CDNA，G5194對G1866進(jìn)行PCR擴(kuò)增，此法去除了內(nèi)G2559G4388的干擾，G14033滿足特殊的實(shí)驗(yàn)要求。CDNA完整序列的獲得對基因結(jié)G7512G451G15519G11345質(zhì)表達(dá)G451基因G2163G14033的研究至關(guān)G18337要。1材料與方法11主要儀器及試劑用品111主要儀器5417R型EPPENDORF離心G7438G708德G3281G709，DYY8C型G8712平電泳儀G708北京市六G980儀器廠G709，DYYIII31A型G8712平電泳槽北京市六G980儀器廠，WFH201B型G13055外G17891G4568G2465G4568儀G708G990G9035G12946科實(shí)業(yè)G7389限G1856G2508G709，干式G5670G9213器G708G7489G5042G3897G11439儀器G7389限G1856G2508G709，TP600型G7811度PCR儀G708G7097本TAKARAG1856G2508G709，HSP250生G2282培養(yǎng)G12677G708G2716G4584G9404市G1008G13864電G4388技術(shù)開發(fā)G7389限G1856G2508G709，GZXGFCG1031011BS電G9921G5670G9213G21735G20130干G10169G12677G708G990G9035G2350G8900實(shí)驗(yàn)G16786備G7389限G1856G2508G709，TU1810G13055外可G16277G2010G1821G1821度G16757G708北京G16901析G17902用儀器G7389限G17143G1231G1856G2508G709，微G8886G9821，HZQCG12366G8680G9032G6403G14645器G708G2716G4584G9404市G1008G13864電G4388技術(shù)開發(fā)G7389限G1856G2508G709。112主要試劑05DEPCG8712G708G1120乙基G9978G11911G18252G11428,DIETHYLPYROCARBONATEG709，1PBSG708G33241000MLG9793菌的去離G4388G8712G1025G2559NACL8G,KCL02G,NA2HPO4115GG2656KH2PO402G，用G11428G18252G16855PH為74，121G263，G9793菌20MING709,RNA抽提變性G909425G異G11839G8708G13985G2164入35MLCBSG13543G1926G9094G70801MOLG812LG7604G8320G18252G19060G13543G1926G9094，PH40，2SDS，1G163G5059基乙醇G709G1025，G18222氯仿G9163G2524G9094G708G18222G297氯仿G297異G6110醇25241G709，異丙醇，75乙醇，DDH2O，DH2O，瓊脂糖，5G104甲醛膠G13543G1926G909401MOLG812MLMOPS，PH70G72740MMOLG812L乙G18252G19060G7275MMOLG812MLEDTA，PH80，37甲醛，G4627G13044，G2559G9352以G19201G70805G173GG812MLG709的01MOLG812L乙G18252G19145溶G9094，RNA抽提用的TAKARA試劑G11430G708G7097本TAKARAG1856G2508G709，EDTAG727瓊脂糖G708G14533G3281G709，MOPSG7593G708MORPHOLINOG709PROPANESULFONICACIDG761，G2465轉(zhuǎn)錄試劑G11430G708G7097本TAKARAG1856G2508G709，PCR擴(kuò)增試劑G11430G708G7097本TAKARAG1856G2508G709，TAEG13543G1926G9094，250BPDNALADDERMARKERG708G7097本TAKARAG1856G2508G709，G1866G4439試劑G3355為G3281產(chǎn)A55113主要用品G72620MLG10639G10839研G11964器，1000MLG19193形G10954，EPG12661，20G173LG451100G173LG4511000G173LG4515000G173LG7550G3848，G12239G9094G7550規(guī)G7696G72620G173L，100G173L，1000G173L,500G173L，G9793菌G10285G12626，G9793菌G9400G13452，G10639G10839G7846，G19230G4388，G980G8437性G6175G3883G451G2487G13629G451G5137G4388，G11719G14533G8616G14406G11411，量G12582G708100MLG709，G12239G9094G12661G70810MLG709，A56A57A58A59A60A61A62A63A64A65A67G9915G749112菌株及培養(yǎng)方式121菌株G11345G14116G6297G4388菌粗毛栓菌G13007導(dǎo)師孫迅于1G28G287年G27G7388G3324G4677G1008G11477G14755G8913市北G18078的G6264G3490G3576G990，G1186G15999G11745G1252的G7484G7653G990G2010離得到的。該G18338生菌株于G2528年G13475G11013G4677G1008G11477科學(xué)G19510生物研究所微生物G2010G12879G4472的G20544啟明先生對G1866進(jìn)行了初步鑒定，定名為TRAMETESGALLICAA68A69A70FR122培養(yǎng)基G726PDA培養(yǎng)基G726去皮土豆，200GG2164G8712煮沸20MIN,冷卻至G4472G9213紗G5079G17819G9400的G9400G9094G709G727葡萄糖，20GG727瓊脂粉，15GG727補(bǔ)G8712至1000ML。按G990述配方配G2058，G9163G2524G3355勻，取10G1022培養(yǎng)G11411G265620支10ML的試G12661，放入G20652壓蒸汽鍋G1025G9793菌G708121，30MING709結(jié)束G2530取出，待培養(yǎng)基冷卻50G2530G2010裝至10G1022培養(yǎng)G11411G265620支試G12661G70810MLG1025，擺斜面，冷卻至G4472G9213G2058G6116固G1319培養(yǎng)基。淺層靜置培養(yǎng)基半纖G13512G13044，3GG727酒G11719G18252G19145，15MGG727MG2SOA71G1037H2O，150MGG727KH2PO4，015GG727酵母粉，30MGG727100G104微量元G13044母G9094，3ML此培養(yǎng)基G7171G726G8616為G726G20652G11911G1314G8706培養(yǎng)基，適于粗毛栓菌G1025G7420聚糖酶的培養(yǎng)。按G990述G8616例準(zhǔn)確稱取各藥品置于50ML的小G9915G7491G1025，然G2530用去離G4388G8712將G17837G1135藥品溶G16311G5194定容150MLG708PH約為65G709，將定容好的培養(yǎng)基溶G9094G3355G2010至G1016G1022250MLG19193形G10954G1025，即G8611G1095450ML。用紗G5079及塑料G13452封好G10954G2487G2530放入G20652壓蒸汽G9793菌鍋G1025G9793菌G708121，30MING709，結(jié)束G2530取出G19193形G10954放至G4472G9213下冷卻。冷卻至35G263左右時(shí)，G3324無菌G4472內(nèi)用接G12193鏟G1186培養(yǎng)了1G19D的G12193G4388培養(yǎng)基G1025挖取三塊米粒大的粗毛栓菌菌G1009G1319G15放入此培養(yǎng)基G1025，即接G12193。然G2530于26G263G5670G9213G12677進(jìn)行培養(yǎng)，G5194于1G19DG2530G6922獲菌G1009G1319。13PCR引物總CDNA的PCR擴(kuò)增的引物G726OLIGODTLACF5GCTCATCGGGCCTAGTCGCA3LACR5TTACTGGTCGTCAGGCGA3以G990引物G7171G11013導(dǎo)師孫迅對G11507菌漆酶G2528工酶基因編碼區(qū)進(jìn)行G2528G9316性G8616較G2656G2010析G2530，根據(jù)編碼區(qū)G1025的G1457G4444序列所G16786G16757的G12628G5194引物G708G11013TAKARAG1856G2508G2524G6116G709。14總RNA的提取與檢測141總RNA的提取使用CHOMCZYNSKIG2656SACCHIA72A73A74所提出的G18252G18222抽提法G5194略作改G2172。1將培養(yǎng)G9094G1025的菌G1009G1319轉(zhuǎn)G12239至三層紗G5079G990，G5194用G990述無菌預(yù)冷PBSG13543G1926G9094G2465復(fù)G1926洗。A76A77A78A79A80A81A82A83A84A85A862取出菌G1009G1319，置于無菌的G3822層G9400G13452G1055間，吸干G3822余的G9094G1319。3用G9793菌G10285G12626迅速將菌G1009G1319大約04G轉(zhuǎn)G12239至G5062G13475DEPCG3800理G17819的20MLG10639G10839研G11964器G1025，G2164入6ML預(yù)冷的RNA抽提裂G16311變性G9094G1025研G11964，直至研G11964G9094呈G3355勻的G8986G9094狀。4G2164入12ML2MOL/L的HACNAACG708PH40G709G13543G1926G9094，G9163G2524G3355勻。5將研G11964G9094轉(zhuǎn)G12239至10只用DEPCG3800理G17819的15MLEPG12661G1025，向G8611只G12661G1025G2164入與樣品G12573G1319積的G708約06MLG709G18222氯仿G9163G2524G9094G708G18222G297氯仿G297異G6110醇25G29724G2971G709，G9163勻，強(qiáng)力G6403G14645G708渦旋G70920SEC，然G2530靜置冰G903210MIN，離心G70812000RPM，4G263，20MIN。6取出G990層G8712G11468至另外5只G13475DEPCG3800理G17819的15MLEPG12661G1025，向G8611只G12661G1025G2164入與樣品G12573G1319積的G708約05MLG709氯仿，離心G70812000RPM，4G263，1015MING709。7取出G990層G8712G11468至另外2只G13475DEPCG3800理G17819的15MLEPG12661G1025，向G8611只G12661G1025G2164入與樣品G12573G1319積的冰冷的異丙醇，G990下G2465轉(zhuǎn)幾G8437G708G9163勻G709，20G263冷凍1020MIN，離心G70812000RPM，4G263，15MING709，棄去G990清夜。8再向G8611G12661G1025G2010別G2164入1ML75的乙醇，離心G70812000RPM，4G263，15MING709，棄凈乙醇，G4472G9213下開蓋靜置510MIN。9G18337復(fù)G17819程G7084G709G7088G709再抽提純G2282G980G8437。10G2164100LDEPCG8712，G5194于20G263下G1457G4396，備用。142總RNA純度與濃度的檢測取25LG990述總RNA的提取G9094G265625ML去離G4388G8712，放入G11719G14533G8616G14406G11411G1025，G9163勻，G2010別G3324260NMG2656280NMG8886長下，用TU1810G13055外可G16277G2010G1821G1821度G16757測量G1866G1821吸G6922值，G17902G17819G16757算A260G2656A280的G8616值測G1866純度G727G5194根據(jù)以下G1856式G16757算RNA樣品濃度，總RNA的濃度G708G/LG709OD260核G18252稀釋倍數(shù)401000。143總RNA的甲醛變性瓊脂糖凝膠電泳A87A88A89稱取04G瓊脂糖于248MLG9793菌G8712G1025，G2164G9921融G2282，冷卻至60G263G2530，G2164入8ML5G104甲醛膠G13543G1926G909401MOL/LMOPS3G708MORPHOLINOG709PROPANESULFONICACID,PH70G72740MMOL/L乙G18252G19060G7275MMOL/LEDTA，PH80G265672ML37甲醛，G9163勻，冷卻至約50G263時(shí)，倒入G2058膠槽G1025，使凝固。取30LRNA樣品與133L5G104甲醛膠電泳G13543G1926G9094，233L37甲醛G2656667L10MOL/L的G4627G13044G9163G2524，65G263，G1457G921315MIN，于冰G9032G1025速冷，G2164入15L10G104G990樣G13543G1926G9094，G9163勻，立即G990樣G708凝膠G5062預(yù)電泳5MING709，G33241G104甲醛膠電泳G13543G1926G9094G1025G5670壓G7085V/CMG709電泳，待G9352G18222G15025染料到達(dá)凝膠G5225部約1/5G3800時(shí)，G1584G8502電泳，將凝膠G9036入G2559G9352乙G19201G70805G/MLG709的01MOL/L乙G18252G19145溶G9094G1025染G1440630MIN，G2530將G1866放入G7389MGCLA90的DHA90OG1025G9036G88853G2010G19059，G7380G2530用WFH201B型G13055外G17891G4568G2465G4568儀G7186G1699，G6305G10043。15RTPCR151逆轉(zhuǎn)錄及PCR利用G7097本TAKARAG1856G2508生產(chǎn)的G2465轉(zhuǎn)錄試劑G11430進(jìn)行逆轉(zhuǎn)錄。逆轉(zhuǎn)錄及PCR所用試劑G11430配A91A92A93A94A95A96A97A98A99A100A101方如下G726G3324G195G80G47G40PG12661G1025G2164入G990表G1025的試劑G708此步實(shí)驗(yàn)樣品RNA為本G4472G1457G4396的RNAG709，于干式G5670G9213器G10255G19G263G1457G9213半小時(shí)。G9213G2656G3332G9163勻G2530，G12257G12257離心，放入G7811度PCR擴(kuò)增儀G1025，進(jìn)行PCRG2465應(yīng)，G7477G1226如下G726G284G2633G80IG81G708預(yù)變性G709G727G284G2633G19SEC，4G28G2635G27G263G708G20104G1022G7811度，G2010別G33241G4515G451G27G45112行G7093G19SEC，72G2631G80IG812G19S，G990述G18613G19G1022G5502G10627G72772G2635G80IG81G7274G263G5670G9213G1457G4396。152PCR擴(kuò)增產(chǎn)物的瓊脂糖凝膠電泳PCRG2465應(yīng)結(jié)束G2530，稱取075G瓊脂糖于50ML05TAEG13543G1926G9094G1025，G2164G9921融G2282，冷卻至約50G263時(shí)，倒入G2058膠槽G1025，使凝固。取25LPCR擴(kuò)增產(chǎn)物與5LG990樣G13543G1926G9094內(nèi)G2559G9352粉G15025G708G15025G13055G14406G709G2656G1120甲G14531G19750G708G15025G14406G709G96，G9163勻，立即G990樣凝膠G5062預(yù)電泳5G80IG81G64G9869G3247G17959，G8611G179591G19G173G47，DNAG80ARKERG708G8611G1022G2164樣G439275LG709G2528時(shí)G990樣于樣品G17959G7061G17805G96，G332405TAE電泳G13543G1926G9094G1025G5670壓G7085V/CMG709電泳。待染料到達(dá)凝膠G5225部約1/5G3800時(shí)，G1584G8502電泳，將凝膠G9036入G2559G9352乙G19201G70805G/MLG709的去離G4388G8712溶G9094G1025染G1440630MIN。G7380G2530用WFH201B型G13055外G17891G4568G2465G4568儀G7186G1699，G6305G10043。2結(jié)果與討論21總RNA電泳結(jié)果G80RNA的質(zhì)量G708G2010G4388的完整性G2656純度G709G3324G19555G2530的G2465轉(zhuǎn)錄G2465應(yīng)G1025，G7171G1927定G14033G2554獲得G1852長CDNA的關(guān)鍵因G13044G1055G980。G11013于RRNAG3324總RNAG1025G2559量G3822G13792G12193G12879G4581，所以根據(jù)G4439G1216的G4506度G2656G2010G4388大小，G17902G17819G4506度G7811度離心G451凝膠電泳G6122離G4388G1144G6454層析G4613可以將G4439G1216G2010離。G4625G12661G80RNAG2010G4388G12193G12879G13333G3822，G1306G7171G1866G2559量G4581，G2010G4388量大小G1075G5468G993G3355G980，所以G3324電泳G3282G16901G990，G5460G5460呈微G5381的G5369G6967形G2010G5079。對新G2058備的總RNAG1025G80RNA質(zhì)量的檢測，G17902G5132G7171G1393G17194凝膠電泳技術(shù)，G17902G17819對RRNA質(zhì)量的G2040G7041G13792確定的。G20652質(zhì)量的總RNAG2058備物的電泳G3282G16901應(yīng)G5415G7171，2G27SRRNA試劑G1319積G708G173G47G7091G19G104G50G81ESTEG83RTPCRG37G88G73G73ERG50G81ESTEG83G40G81G75AG81CERSOLG88TIOG81DNTPG48IG91TG88REG7081G19G80G48EACG75G709RNA102A103A104G44G81G75IBITORG7084G19G753G812G173G744G709PRIG80ESCRIG83TA105A106RTASEG73OR1STEG83TAKARAEXTAQA107A108HS5G56G812G173G47G990G9228特異性引物G7082G19G173G48G709下G9228特異性引物G7082G19G173G48G709實(shí)驗(yàn)樣品RNA102A103A104FREEDG43A109G501G192421222273A110A111A112A113A114A115A116A98A99A100A101G26561G27SRRNA的G5114形整G21796，G1000無G14085G4626G6122G5369G6967G10628G16949G727G7389時(shí)，2G27SRRNAG5114的G1154度可達(dá)到1G27SRRNA的2倍。G3324總RNA的G2058備G17819程G1025，如果G11013于G6817作G993G5415G6122G11013于RNASE的G8757染G13792G17908G61162G27SRRNAG26561G27SRRNA部G2010G19489G16311，G18039G1052，G3324電泳G3282G16901G990，G4439G1216的G5114形G4613G1262出G10628G1017G18337G6314G4626G6122G5369G6967G10628G16949，G10990至G993G6116G5114形，G980G7098出G10628G17837G12193G5785G1929，G4613G16840明G80RNA的質(zhì)量G1075G5062G13475G8821G7389G1457G16789了，即G993G14033用于RTPCR研究工作。因?yàn)镚3324總RNAG1025，G80RNA應(yīng)G5415G7171G1288G19555G115402G27SRRNAG26561G27SRRNA的G19489G16311G13792G19489G16311的，尤G1866G7171G11013于RNASE引G17227的G19489G16311作用G7368G7171如此A117A118A119。本G8437實(shí)驗(yàn)結(jié)果電泳G5194G7422出G10628理G5831的G3282G16901，G2419因G2010析可G14033主要G7171G11013于RNA樣品G3324抽提G17819程G1025G3324RNA102A103A104作用下裂G16311G6122勻G8986G3800理G993G5455G5225G6122G13785G7171總RNA量G3838G4581所致。22總RNA濃度與純度G332426G19G81G80G8886長下，1G50D值的G1821G4506度G11468G5415于G2345G19154RNA濃度的4G19G173GG18G80G47，所以本實(shí)驗(yàn)所提總RNA樣品的濃度為G726G50DA109A120A121G104核G18252稀釋的倍數(shù)G1044G19G1141G19G19G19G32G1946G104G70825G19G19G1825G709G1044G19G181G19G19G19G32G191G274GG18G47，G3324G2465轉(zhuǎn)錄時(shí)，4G19G47G2465轉(zhuǎn)錄G9094G1025的總RNA的濃度為G191G274G10425G184G19G32G19115GG18G47。測得G50DA109A120A121，G50DA109A122A121值G2010別為G1946G2656G1932G15所以G50DA109A120A121G18G50DA109A122A121的G8616值為144，G16840明總RNA純度較G1314，G13475G2010析G2419因G5468G7389可G14033G7171因?yàn)闃悠稧1025G9163G7389G18222G2656G15519G11345質(zhì)的G13548G6937。23PCR擴(kuò)增結(jié)果G11013于G1055前RNA提純效果G993理G5831，所以G3324進(jìn)行RTPCRG17819程G1025所用的RNA為本實(shí)驗(yàn)G4472G1457G4396的RNA，PCR擴(kuò)增結(jié)果如下G726A123A124A125A126A127A128A129A130A131A132A133A134A135A136A137A138A139A140A141A142A143A144A145A140A141A146A145A144A147A140A141A146A146A144A142A140A141A146A143A144如G3282G1025可以G11487出，G3247G1022G9213度G7811度G1025，4G179595G27G263時(shí)G7477G5114G7380為明G7186，G16789明G33245G27G263時(shí)PCR擴(kuò)增效果G7380好。擴(kuò)增出G7481的G1016G7477CDNA長度G980G7477約為145G19BG83左右，另G980G7477約為12G19G19BG83A110A111A112A113A114A115A116A98A99A100A101左右。RNAG8616DNAG6947G5875，G7143受G19489G16311，G13792RNA酶卻G8616DNA酶G2010G5079G7368G5203G8879，G8975力G7368強(qiáng)，G980G14336煮沸RNA酶510MIN，G186675的G8975性G1185然G8543G4396，所以G3324RNA的提取G1055前，必須對所要用的實(shí)驗(yàn)儀器