Maitiro ekugadzirira masiraidhi e comet assay
The thecomet assay. Kugadzirira kukwirira - HunhuComet assay slidein inhanho yakaoma inosarudza kuvimbika uye kubereka kwemhedzisiro yemubhadharo. Chinyorwa ichi chinotarisira kupa nhungamiro yakazara pakugadzirira masiraidhi kuti ivhu asingaguti, kunhuhwirira kunonzwisisika kubva kunyanzvi mumunda. Tichafukidza nhanho imwe neimwe yakakosha, kubva pasero kusarudza kududzirwa kwedata, ichivimbisa iwe kuti uwane mhedzisiro yakanakisa.Nhanganyaya yekunyora Assay uye kukosha kwayo
● Ongorora yeComet Assay
Iyo COMET leasay, yakagadziridzwa mukupera kwe1980s, chishandiso chine simba chekuongorora dhizaini DNard By of offary eukaryotic masero. Iyo inoshandiswa zvakanyanya muminda yakadai sekuongororwa kweiyo genity yekuyedza, iyo molecular epidemiology, uye yezvakatipoteredza biomonitoring. Iyo nheyo yeiyo comet assay inosanganisira masero emabheji muAgarose, marara kuti abvise membran nemapuroteni, uye wozotarisa maElectrophoresis. Mufananidzo wakakonzerwa, wakafanana neye comet, inobvumira kuwanda kweDNA DNA.● Zvishandiso muDNA Dair Deatment
Comet assays anonyanya kukosha mukuona DNA kukuvadzwa kwakakonzerwa nemamiriro ekunze emamiriro ekunze, radiation, uye mishonga yemishonga. Ivo zvakare vanotamba basa rakakosha mukuongorora kushanda kweDNA rekugadzirisa matanho mumhando dzakasiyana siyana. Vatsvagiri vanoshandisa comet assays yekudzidza cancer, kuchembera, uye zvimwe zvirwere zvakabatana neDNA Dair.● Kukosha mukuedzwa kweGenoticity
Iyo yekuzvidzora uye yekutsvaga kukosha kweiyo comet ass haigone kuve pasi. Ivo vanoshanda sechinhu chakakosha chekutarisa chishandiso chekuzivisa zvinogona kuitika makomputa. Mhedzisiro kubva kune aya maStings inogona kupesvedzera chisarudzo - kugadzira maitiro mukubudirira zvinodhaka uye kwekuchengetedza kwezvakatipoteredza ongororo.Kusarudzwa kwemasero akakodzera e comet assay
● Mhando dzemasero anoshandiswa
Sarudzo yemasero inogona kukanganisa zvakanyanya mhedzisiro yeiyo comet assay. Masero anowanzo shandiswa anosanganisira lymphocytte, maseru ebhisiki, uye akasiyana masekonzi ezvinyorwa mitsara. Vanotsvagurudza vanowanzo sarudza marudzi emusero zvichienderana nezvinangwa zvekudzidza uye kugadzikana kwekuwana samples.● Kufungisisa kwechisarudzo cheter Cell
Kana uchisarudza masero, funga nezvezvinhu zvakadai sekuwanika kwesero, kunzwa kweDNA kukuvara, uye kukosha kumubvunzo wekutsvagisa. Semuenzaniso, lymphocyte inowanikwa nyore kubva paropa uye ine hanya neiyo genotic vamiriri, kuvaita sarudzo yakanaka kwazvo yezvidzidzo zvakawanda.● Umanikidza mhedzisiro fungidziro
Mavambo uye mamiriro emasero anogona kukanganisa iyo baseline mwero weDNA DNA uye iyo sero mhinduro kurapwa. Naizvozvo, kusarudzika sero kusarudzwa uye kubata protocol kwakakosha kuti uwane yakavimbika uye kubereka mibairo mu comet assays.Kugadzirira kweAgarose Gels
● Mhando dzeAgarose kushandisa
Kusarudza iyo chaiyo agarose yakakosha kuti uvimbe neye comet assa. Low LELTING PONEST AGAROSE AGAROSE AGAROSE ANOGONESESWA KUTI ISHE ISSIFITION SUPLY uye kuderedza DNA kukuvara panguva yekugadzirisa. Yakakwira - mhando Agarose kubva kune vanokurumbira vatengesi, vakadai sevaya vanobva kune yakavimbika comet Assay Slide mutengesi kana comet Assay Slidecurer, inokurudzirwa.● Agarose Kusimbisa uye Kugadzirira Matanho
Kazhinji, imwe 1% yakaderera yekunyungudza polting agarose kusungwa kunoshandiswa. Nyungudika iyo agarose mune yakakodzera buffer, kupisa iyo kusvika yakanyungudutswa yakanyungudutswa, uye wozotonhorera kune tembiricha yakachengeteka yekumanikidza. Ita shuwa kuti agarose akasununguka kubva kune mabhuru uye marara kuti adzivirire madhiri mune lefays.● Kuvhiringidza kunaka kweGel
Unhu hwegomba agarose hunogona kupesvedzera kunzwa kwega uye kubereka. Kubata kwakakodzera uye maitiro ekugadzirira kwakakosha kubereka homogeneus gel ine zvinowanzoitika zvivakwa zvakasiyana. Izvi zvinovimbisa kuti kuumbwa kwezvinhu uye kuumbwa kweruzivo uye kuona kwatunifomu.Kuparara kwemasero uye masero emabheti mu agarose
● Kugadzirira iyo cell kumiswa
Tanga nekuwana imwe chete - cell kumiswa. Kwemasero ezvakagadziriswa, izvi zvinosanganisira trypsinization, ichiteverwa nekusarerekera nekugeza. Zvemasero ekutanga kunge lymphocyte, centrifugation uye yekuzorora mune yakakodzera buffer inodiwa. Shandisa hemecometer yekuve nechokwadi chekusangana kwesero kweanenge 1 x 10 ... 5 masero / ml.● Kubiridzira masero mune yakaderera yekunyungudika pollo
Sanganisa iyo cell kumiswa ne pre - inodziya yakaderera yekunyungudza polting agarose kune chiyero chinovimbisa kugoverwa kwemasero. Kurumidza pipette musanganiswa wepamberi pane pre - yakafukidzwa comet assay masiraidhi, iyo inogona kutengwa kubva kune comet assay slide fekitori kana mutengesi akavimbika. Shandisa kuunganidza kuti uparadzire iyo agarose nenzira kwayo uye kudzivirira mhepo mabhuru.● Kuparadzira masero pamasiraidhi
Kana iyo agarose ikapararira, bvumira iyo slides kuti itonhore uye inosimbisa pane inotonhora nzvimbo kana nzvimbo yefiriji. Iyi nhanho inofanira kuitwa zvinobudirira kudzivirira iyo agarose kubva pakuisa zvisina kukodzera, izvo zvinogona kukanganisa kuoneka kwemazita panguva yekuongorora.Lyns yemasero pamasiraidhi
● Lyns Supindura Kuumbwa
Solis Solised kazhinji ine manyorerwo enyasha uye undergents, seNacl, Edta, uye zvinobudirira Lyre cell uye bvisa membranes uye mapuroteni uchisiya dna.● Lotss maitiro uye mamiriro
Nyora masiraidhi muLaysis Solution uye Incubate pa 4 ° C kweinenge 1 awa, kunyangwe usiku hwehusiku hwekuwedzera kazhinji. Ita shuwa tangi yeLysis yakafukidzwa kuti udzivise kubuda uye kusvibiswa. Mamiriro ezvinhu eiyo lyiss danho haana kuderera kubvisa cellular cellular cellular cellulars pasina kushatisa DNA.● Kukosha kweiyo yakazara cell loysis
Nyowani isina kukwana inogona kukonzera kusarongeka kwekuumbwa uye isina kuvimbika data. Naizvozvo, zvakakosha kutevedzera iyo lysis protocol zvine musoro, kuona kuti mhinduro inopinda mukati mezvikamu zvese zveiyo slide uye nguva yekutsvedza iyo yakakwana yekukwana kwekuzadza cell.Alkaline Unwinging uye Electrophoresis mamiriro
● Kugadzirira mhinduro yeAlkaline
Iyo alkaline solution, kazhinji inosanganiswa nezvaNao uye edta, inoshandiswa kusunungura DNA. Kugadzirira kwayo kunofanirwa kuitwa nokungwarira, kuve nechokwadi chechokwadi ph uye molarity. Nyango gadzirira mhinduro yekuchengetedza maitiro ayo.● Alkaline Unwinging Zvakakosha uye Nguva
Incubate masiraidhi muAlkaline Solution, kazhinji kwemakumi maviri - maminitsi makumi mana, kubvumira iyo DNA strANDS kusunungura. Iyi nhanho yakakosha yekuona imwe chete - Strand break uye Alkali - Nziyo Nyowani. Edza nguva isingazivikanwe zvichienderana neyako chaiyo protocol uye sero mhando.● Electrophoresis Setup uye Paramita
Seta iyo electrophoresis tangi ine inotonhora alkaline electrophoresis buffer. Isa masiraidhi ari mutangi, achivimbisa kuti vakanyatsoiswa. Nyorera iyo inowirirana voltage (kazhinji ichipoterera 25 v) ye20 - maminetsi makumi matatu. Mamiriro eElectrophoresis, kusanganisira voltage uye nguva, inofanira kuve yakagadzirirwa kuzadzisa iyo yakanyanya kugadziriswa kweDNA kutama.Kusarerekera uye kusanganisa maitiro
● Kusarerekera Buffer Kugadzirira uye Kushandisa
Mushure mekunge Electrophoresis, nyadzisira masiraidhi ekumisa maitiro uye kusimbisa DNA. Kusarerekera kweraramu Nyoro geza masiraidhi mune ino buffer kanoverengeka kubvisa zvese zvinongedzo zveAlkaline Solution.● Kubata nzira
Stain yetratelized slides ine fluorescent DNA - inosunga dhayi, yakadai seEtifium Bromide kana SyBR Green. Trifiium Bromide, nepo Classic, inoda kubata nekuwaridza nekuda kwezvinhu zvayo mumagegenic. SYBR Green inopa imwe nzira yakachengeteka neyakanyanya kunzwa. Intubate masiraidhi mumamiriro ekusagadzikana kwenguva yakakodzera (kazhinji 15 - maminetsi makumi matatu) murima.● Kuvonekwa kweDNA COCET
Mushure mekusunga, geza masiraidhi zvinyoro-nyoro kuti ubvise zvakawandisa banga. Wongorora masiraidhi pasi peiyo fluorescence microscope. Hunhu hwekusimbisa uye microscope resolution akakosha kuti uwane mifananidzo yakajeka yemazi.Microscopy uye mufananidzo ongororo
● Kusarudza iyo microscope chaiyo
Iyo fluorescence yemicroscope yakashongedzwa neyakagadzirirwa faira yakafanira yekuona yekuona DNA. Sarudza microscope nehurongwa hwepamusoro uye kamera system inokwanisa kutora mifananidzo yakadzama yemazi.● Kutapa Kukwirira - Mhando Mifananidzo
Tora mifananidzo yemashanu inosvika makumi mashanu emapundu pane slide yekuve nechokwadi chekukosha. Ive nechokwadi chekuti kunyange kuvhenekerwa uye kutarisa kuti udzivise madhiri. Zvakakodzera Calibration yeMicroscope uye kamera marongero inogona kuwedzera zvakanyanya mufananidzo mhando.● Software yekuongorora comet
Zvirongwa zvakati wandei zviripo zvinowanikwa zvekuongorora comet, zvakadai se canp. Izvi zvirongwa zviyero senge muswe urefu, kusungwa muswe, uye muswe nguva, zvinokosheswa nekuda kwekutanga kweDNA kukuvara.Kugadzirisa matambudziko zvakajairika
● Matambudziko Angori
Nyaya dzakajairika muComet assays inosanganisira yakakwira backgrescence, yakaderera comet fomu, uye isingaenzaniswi mhedzisiro pakati pemasiraidhi. Matambudziko aya anogona kubva kune akasiyana siyana, kusanganisira mushe masero kubata, Suboptimal Gel Kugadzirira, uye Rwiyo rusina kunaka kana mamiriro eElekituru.● Mhinduro uye matanho ekudzivirira
Kuti uderedze yakakwirira yekumashure fluorescence, ita shuwa kuti vese reagents uye gels vakasununguka kubva kune zvinosvibisa. Zvekuumbwa zvakaderera comet, simbisa kuti cell viability yakakwira, uye mamiriro ekunyora ane ruzivo. Kunyanya kugadzirira kugadzirira uye kunyorwadza protocol kuomerera matanho akakosha ekudzivirira.● Kuverengera kubereka uye kurongeka
Kuwana kubereka mu comet assays inoda kuomarara protocolization statomisation uye kugara uchisimbiswa kwedanho rega rega. Nguva dzose calibrate michina, shandisa yakakwira - mhando regents, uye chengetedza zvinyorwa zvakadzama zvemamiriro ese ekuedza kuti ave nechokwadi chechokwadi.Dudziro yedata uye kubvuma mhinduro
● Kuongorora comet muswe paramita
Key parameter yekudzora DNA kukuvadza kunosanganisira kureba muswe, iyo inoratidza huwandu hweDNA kutama, uye kusungwa muswe, izvo zvinoreva huwandu hweDNA mumuswe. Muorivhi muswe wenguva, kuyerwa kwakaita, kunowanzoshandiswawo.● Kufungisisa kwehuwandu mukubika data
Kuongororwa kwakakwana kwehuwandu kunokosha kududzirwa data. Shandisa yakakodzera nhamba yehuwandu kuti enzanise iyo DNA kukuvara mazinga pakati pemapoka akasiyana ekuenzanisira. Ita shuwa kuti data kubva kune akawanda masiraidhi uye kuedza kunonunurwa kuti uverenge zvisizvo.● Kuburitsa uye kududzira zvakawanikwa
Ratidza mhedzisiro yako nenzira yakajeka uye yakajeka, uchishandisa girafu uye matafura kuratidza zvinhu zvakakosha. Kurukura zvinorehwa neDNA yekukuvadzwa kweDNA yakacherechedzwa uye zvinokwanisika zvinokanganisa pazvinhu zvevabheya pasi pekudzidza.Mhedziso: Kuwana Yakakwira - Hunhu Hwakanaka Comet Assay Slides
Kugadzirira kukwirira - Hunhu Comet Assay Slides inosanganisira kunyatsofungisisa kusarudzwa kwesero, Agarose Gel Kugadzirira, Mamiriro ezvinhu emamiriro ezvinhu, magetsi, uye kuongorora mifananidzo. Nhanho imwe neimwe inofanirwa kuve yakanyatsogadzirirwa uye yakaenzana kuti ive nechokwadi chakavimbika uye chakabudirira mhedzisiro. Kubatana neCommunable Comet Assay Slide Spotliers uye Vagadziri vanogona kuwedzera kunaka kwesimba rako nekupa yakakwira - Zvishandiso zveGiredhi uye technical tsigiro.● NezveIfaseBiosciences
Musoro wakadzika muNorth Wales, Pennsylvania, Pefese Boscience is "nyanzvi ye" nyanzvi, uye inokwirisa " Kuverengera ruzivo rwakakura uye shungu dzekutsvaga kwesainzi, timu yedu yesainzi yevanoona nyanzvi yezvinhu zvakasarudzika zvekutangisa kune dzinza Kutsvaga r & d yakanaka ye "Runyanzvi RegentsKutumira Nguva: 2024 - 08 - 28 14:05:21