Methylene blue yekuisa ruvara muDNA neRNA: kushandiswa mumajeli nemamembrane

Mudzidzi anoita electrophoresis mujeli reagarose, oriisa ruvara nemethylene blue, asi haaoni chinhu panzvimbo yaaitarisira bhandi rinopenya. Pane imwe nzvimbo yekushandira iri pedyo, muongorori ane dhigirii rechiremba anoisa ruvara pamembrane yeNorthern achishandisa bhodhoro rimwe chete rebhuruu, obva awana mabhendi eribosomal akajeka mumaminitsi matatu. Mudzidzi anofunga kuti jeli rakundikana. Jeli harina kukundikana. Mabasa acho maviri aive akasiyana, uye dhayi rimwe chete rinoapindura nekugona kwakasiyana kuona zvinhu zvishoma, ruvara rwemashure rwakasiyana, nemitemo yakasiyana yezvinoitwa zvinotevera.

Kuisa ruvara muDNA nemethylene blue uye kuisa ruvara muRNA nemethylene blue zvinoshandisa kemisitiri imwe chete asi zvinangwa zvakasiyana. Mujeli, dhayi rinoratidza huwandu hwenucleic acid huri mukati mematrix ine maburi madiki. Pamembrane, rinoratidza kana kutamisa kwakashanda uye kana zvakaiswa mumigero zvakaenzana. Kana musiyano uyu wava pachena, maitiro, miganhu yekuona, nemiganhu yekuenderana zvinobva zvanzwisisika.

Mhinduro pfupi

Methylene blue idhayi rine chimiro chakati sandara uye chaji iri positive, rinosunga nucleic acid pasina covalent bonds uye nenzira inogona kudzoserwa shure. Rinoshandisa musanganiswa wekukwezvana kwemachaji kune phosphate, kupinda pakati pemapeya emabhesi, uye kubatana mumukoto mudiki. Kukwanisa kudzoserwa shure uku ndiko kwakakosha. Mabhendi anogona kuiswa ruvara, oonekwa nechiedza chinooneka, orekodhwa, ozobviswa ruvara kana kucheneswa kuti ashandiswe mukugadzira makopi eDNA, kuhybridiza, kana kuongororwa zvakare neprobe.

Pakuisa ruvara ne methylene blue mujeli reagarose rine DNA yetambo mbiri, musanganiswa unokurumidza unoshandiswa zvakajairika i0.02 percent w/v (200 micrograms per mL) mumvura, kuisa ruvara kwemaminitsi angangoita 15 pamwe nekubvisa ruvara nemvura kwemaminitsi angangoita 15, zvichionekwa pabhokisi rechiedza chichena. Pamamembrane, musanganiswa wekare weNorthern blot unoshandisa dhayi re0.02 kusvika 0.04 percent musodium acetate pedyo nepH 5.2, kuisa ruvara kwemaminitsi mashoma mushure mekutamisa nekusungirira panzvimbo, wozosukurudza nemvura uye wobvisa ruvara rwese usati waita hybridization. Kugona kuona zvinhu zvishoma kwakaderera: makumi kusvika kumazana emananograms pabhandi rejeli kana mamiriro akanaka, uye magwaro anopa fungidziro yakangwarira anoti 0.5 kusvika 1 microgram. Huwandu uhwu hwakakwira nezvikamu zvakawanda zvegumi pane hunodiwa ne ethidium bromide nemadhayi eSYBR. Pamamembrane hapana muganhu mumwe chete unobatsira wekuona transcript imwe, nokuti dhayi rinonyanya kuratidza ribosomal RNA yakawanda pamwe nehuwandu hwese hwezvinhu zvakaiswa. Usatsiva musanganiswa upi zvawo ne solution yechigadzirwa chekuwedzera chinotengeswa muzvitoro. Nzira dzakasimbiswa dzinotarisira dhayi regiredhi rekuongorora, mabuffer akanyatsotsanangurwa, uye kubata kusina RNase panoshandiswa RNA.

Kusunga kunoshanda sei, uye kunotsausa papi

Muenzaniso unobatsira une zvikamu zvitatu. Chekutanga, chimiro chemhete chine chaji iri positive chinokwezvwa nemusana wephosphate une chaji iri negative. Ongororo dzespectroscopy dzinoona kuti kukwezvana kwemachaji uku ndiko kunonyanya kukosha kana munyu uri mushoma, asi mienzaniso yekombiyuta nekuongorora kufamba kwemamorekuru zvinoratidza kuti kupinda pakati pemapeya emabhesi nekusunga mumukoto mudiki zvinoramba zviri nzira dzakadzikama dzeDNA yetambo mbiri, sezvakapfupikiswa muongororo yespectroscopy yekusunga kwe methylene blue kuDNA. Chechipiri, pane kumwe kufarira nzvimbo dzeGC: mienzaniso yekombiyuta inofarira matanho eCpG neGpC, uye miedzo inoratidza kubatana kunonyanya kuitika pamatambo maviri ane GC yakawanda mutsvakurudzo yezvinofarirwa nemethylene blue pakupinda pakati pemapeya emabhesi, asi matambo maviri eAT anosungawo. Kuisa ruvara kwakajairika hakusarudzi kutevedzana kwemabhesi kwakati. Chechitatu, DNA yetambo imwe neRNA zvinonyanya kusunga kuburikidza nekukwezvana kwemachaji uye kurongedzana nemabhesi anosvikika nenzvimbo dzine matambo maviri, ndosaka kusimba kweruvara kuchisiyana nehurefu, kutevedzana kwemabhesi, uye chimiro chinobva pakupetana.

Pane zvinobva pazviri zviviri. Nokuti kusunga hakuitwi nema covalent bonds, dhayi rinobuda pakusukwa. Mabhendi epolyacrylamide anokurumidza kupera ruvara kunze kwekunge asungirirwa nemaune, uye pane nzira yakatsaurirwa yekusungirira zvachose nokuti kuisa ruvara kwakajairika ndekwenguva pfupi, sezvinotsanangurwa naSoto naDraper pamajeli ePAGE. Nokuti dhayi rinokurudzira maitiro emakemikari kana ravhenekerwa, kuvhenekera kwenguva refu nechiedza chakajeka DNA yakaiswa ruvara paine oxygen kunogona kuita oxidation yeguanine uye pakupedzisira kutsemura tambo, maitiro anotsanangurwa mutsvakurudzo yeoxidation yeguanine inokurudzirwa nemethylene blue. Kutarisa kwenguva pfupi nechiedza chichena kunodzivisa kukuvara kweUV kunozivikanwa munzira dzekare dze ethidium bromide, uye mukana iwoyo ndewechokwadi pakudzosa DNA kuti ishandiswe zvakare. Chiedza chinooneka hachinyanyi kukuvadza, asi hachisi chisina njodzi. Vhenekera kwenguva pfupi kana DNA ichizoshandiswa kugadzira makopi. Kuti uzive zvakawanda nezvekemisitiri yechiedza iri shure kweyambiro iyi, ona nzira mbiri mukemisitiri yechiedza yemethylene blue. Kuti uzive nezvemhando mbiri dzinopindurana kuburikidza neredox uye mamiriro echaji anotsanangura kuti nei mhando ine oxidation ichisunga asi mhando yakaderedzwa isingasungi, ona kemisitiri yechimiro nehunhu hwemethylene blue.

Basa rekutanga: kuisa ruvara mukati memajeli

Kuisa ruvara mujeli kunopindura mubvunzo mumwe: pane nucleic acid yakakwana here mubhandi iri kuti ionekwe neziso. DNA yetambo mbiri muagarose ndiyo yakanyatsotsvakurudzwa. Nzira yekutanga yemethylene blue mumajeli yaFlores nevamwe yakasimbisa nzira inokurumidza ye0.02 percent paagarose ye1 percent uye yakaratidza kuti DNA yaigona kudzoserwa uye kushandiswa kugadzira makopi zvinobudirira mushure mezvo. Maitiro mamwe chete emutengesi anoonekwa papeji rechigadzirwa chemethylene blue reIBI Scientific, rinoratidza musanganiswa wemamembrane we0.04 percent pamwe chete nenzira inokurumidza yemajeli.

Matanho ekushanda:

1. Ita electrophoresis mujeli sezvenguva dzose. Isa ruvara mushure meelectrophoresis, kwete ichiri kuitika, kunze kwekunge kutamisa kana kupatsanura zvakanyatsosimbiswa panzira yakagadziridzwa.
2. Nyudza mu methylene blue ye0.02 percent w/v mumvura yakadistillwa, uchizunza zvinyoronyoro, kwemaminitsi angangoita 15.
3. Bvisa ruvara nekuchinja mvura kakawanda, kwemaminitsi angangoita 15 ose pamwe chete, kusvikira ruvara rwemashure rwematrix rwadzikira uye mabhendi ava kuoneka zvakajeka.
4. Tora mufananidzo nokukurumidza pamudziyo unovhenekera nechiedza chichena.

Agarose nepolyacrylamide zvinoshanda zvakasiyana. Agarose inochengeta dhayi mumatrix, saka kubvisa ruvara ndiro danho rinononoka. Majeli matete epolyacrylamide anokurumidza kuiswa ruvara asi anokurumidzawo kurasikirwa narwo, izvo zvinobatsira pakucheka zvikamu zvekuzoshandisa asi zvichikanganisa marekodhi ekuchengetera. Maoligonucleotide akagadzirwa anogona kuwanikwa mupolyacrylamide uchishandisa musanganiswa mumwe chete we0.02 percent mumaminitsi 20 kusvika 30, uchisuka nemvura kakawanda, uchitevera nzira dzePAGE dzakagadzwa dzemhando yeCurrent Protocols. Nzira dzine dhayi shoma zvikuru, pedyo ne0.0002 percent muTBE yakanyungudutswa zvakanyanya, dzakashumwa dzeoligo dzinogadzirirwa kuzoshandiswa kwemaawa akati wandei, asi humbowo hwadzo hahuna kusimba zvakafanana.

Musiyano pakati peDNA neRNA wakakosha. DNA yetambo mbiri inopa kusunga kwakasimba zvikuru kunoenderana nechimiro chayo. RNA neDNA yetambo imwe zvinoiswa ruvara, asi zvisina kusimba uye zvisinganyatsofanotaurwi. Pamajeli eRNA eformaldehyde agarose, marabhoritari mazhinji haaise ruvara rwebhuruu mujeli asi anotanga atamisa RNA kuenda pamembrane, ozoisa ruvara ipapo. Kuchinja uku kunotsiva chiratidzo chisina kusimba mujeli nechiratidzo chakajeka pamembrane pamwe nerekodhi rezvakaiswa.

Basa rechipiri: kuisa ruvara pamamembrane

Kuisa ruvara pamembrane kunopindura mumwe mubvunzo: kutamisa kwakashanda here, uye migero inogona kuenzaniswa here. Kushandiswa kwekare ndiko kuisa ruvara kweNorthern blot kwaHerrin naSchmidt kunogona kudzoserwa shure. Pakutanga kwaishandisa dhayi re0.04 percent mu50 mM sodium acetate papH 5.0 kwemaminitsi 2 kusvika 3, kuchiteverwa nekusukurudza nemvura; raibviswa neSDS yakanyungudutswa zvakanyanya muSSPE hybridization isati yaitwa.

Nzira yemazuva ano inoshandiswa zvakanyanya inotevera matanho aya:

1. Tamisa RNA kana DNA yakaparadzaniswa tambo kuenda panylon kana nitrocellulose, wobva waisungirira panzvimbo nekubatanidza uchishandisa UV kana nekupisa kuitira kuti sampuli isarasike panguva yekuisa ruvara.
2. Hazvisungirwi asi zvinowanzoitwa: nyorovesa membrane muacetic acid ye5 percent kwemaminitsi angangoita 15.
3. Isa ruvara kwemaminitsi 5 kusvika 10 mu methylene blue ye0.02 kusvika 0.04 percent iri mu0.3 kusvika 0.5 M sodium acetate pedyo nepH 5.2.
4. Sukurudza wobvisa ruvara mumvura kwemaminitsi 5 kusvika 15, rekodha, wobva wabvisa dhayi rasara usati waita hybridization.

Nylon ine chaji iri positive ndiyo inonyanya kushandiswa mubasa reNorthern. Nitrocellulose inoshandawo nematanho mamwe chete eacetic acid pamwe neacetate. Mufananidzo unoshandiswa kusimbisa kutamisa, kuenzanisa mabhendi e28S ne18S (kana 23S ne16S paRNA yemabhakitiriya), kumaka maladder nezviyero, uye kuchengeta rekodhi rinofungidzira rekuenzanisa huwandu hwese hweRNA. Nzira dzeNorthern dzakabudiswa dzinoyera mabhendi eribosomal akaiswa ruvara kuti dzitarise huwandu hwakaiswa, kusanganisira tsvakurudzo yesainzi yemukaka yakashandisa mabhendi eribosomal akaiswa ruvara nemethylene blue pakuenzanisa huwandu. Mamembrane eSouthern anogona kuonekwa nenzira imwe chete, asi dhayi rinozivikanwa zvakanyanya sekutarisa huwandu hwakaiswa nekutamisa muNorthern, uye DNA yeSouthern inoparadzaniswa tambo isati yatamiswa, saka manhamba ekugona kuona mujeli haashandi ipapo.

Miganhu yekuona neruvara rwemashure: panokundwa nebhuruu

Kuenzanisa kuri pasi apa kunopa fungidziro nemaune. Kushanda kwedhayi kunoenderana nechikamu chejeli, upamhi hwebhandi, midziyo yekuona, uye kunyatsotevedza kubvisa ruvara. Tora nhamba imwe neimwe sechiratidzo chehukuru hwehuwandu, kwete sechiyero chakatarwa.

Kudzora ruvara rwemashure ndiko kunosarudza kana chiratidzo chisina kusimba chichigona kushandiswa. Dhayi risina kusunga rine ruvara rwakasimba, saka musiyano unoonekwa unobva pakubvisa dhayi risina kusunga pasina kubvisa rakasunga. Dhayi rakawanda mumusanganiswa hariwanzo wedzera kugona kuona zvinhu zvishoma. Rinonyanya kuwedzera nguva yekubvisa ruvara. Edza nhevedzano yehuwandu hunoderedzwa pajeli, pamembrane, uye pakamera zvauchashandisa, uye shandisa chete chikamu umo simba rose rechiratidzo rinoramba richienderana nehuwandu hwakaiswa. Methylene blue inobatsira pakuenzanisa huwandu hwakaiswa nenzira inopa fungidziro yenhamba. Haisi bvunzo yekuyera huwandu pamusoro pemhando yakafara yezviyero nokuti kugona kwemembrane kubata zvinhu, kubudirira kwekutamisa, kuzara, uye kubvisa ruvara zvinosiyana kubva pamugero mumwe kuenda kune mumwe.

Miganhu yekuenderana: chii chinoramba chichishanda mushure mekuiswa ruvara

Kugadzira makopi eDNA mushure mekuiswa ruvara rwebhuruu kwakaratidzwa. Flores nevamwe vakacheka mabhendi eagarose akaiswa ruvara, vakadzosa DNA, vakaibatanidza uye vakaiisa mumasero, vachishuma kugadzirwa kwemakopi kuri nani pane kwakaitwa mushure me ethidium bromide pamwe neUV. Chikonzero chingangove kuderera kwekukuvadzwa nechiedza, kwete kubatsira kunobva padhayi pacharo. Ita kuti kuratidzwa kuchiedza chichena kuve kupfupi uye chenesa DNA yadzoserwa nenzira yakajairika yekuibvisa mujeli.

Humbowo hwePCR nekuverenga kutevedzana kwemabhesi hushoma. Dhayi rasara rinosunga nucleic acid, saka bvisa ruvara uye chenesa usati waita matanho anoshandisa maenzyme. DNA yakanyatsocheneswa kubva mujeli rine bhuruu inotarisirwa kushanda, asi huwandu hwedhayi rinotakurika richienda kumatanho anotevera hunotsungirirwa nemapolymerase akasiyana nemakemisitiri ekuverenga kutevedzana hahuna kunyatsoyerwa. Usazvifungidzira.

Hybridization yeblot inoenderana nazvo kana kurongeka kwematanho kuchinyatsotevedzwa: tamisa, sungirira panzvimbo, isa ruvara, rekodha, bvisa ruvara, hybridiza. Kurongeka ikoko ndiko kwakagadzirwa naHerrin naSchmidt. Kuisa ruvara usati watamisa, kana kusvetuka kubvisa ruvara, inzira yakasiyana uye inoda kusimbiswa kwayo pachayo. Mushure mekubvisa dhayi, kubvisa probe nekuisa imwe probe patsva kunoenderera sezvenguva dzose. Kuti uzive nezvekuisa ruvara pamasero uchishandisa mhuri imwe chete yemadhayi uye kuti nei mafungiro ekushanda nemafirimu akasungirirwa asingashandi panzira dzemasero mapenyu, ona atlasi yekuisa ruvara mukuongorora nemicroscope.

Kucheka mabhendi kunoshanda paagarose yekugadzirira zvinhu zvekuzoshandisa uye paoligo dzePAGE dzakawanikwa nebhuruu, chero bedzi nzira yekusungirira zvachose isingashandiswi. Kuburitsa nemagetsi mushure mekubvisa ruvara kunonzwisisika mukemisitiri yemusungi anogona kubviswa zvakare, asi mashoko akabva mumiedzo inodzorwa ehuwandu hunodzoserwa achinyatsotaura nezvemethylene blue mashoma. Bvisa munyu kana kuita kuti zvinhu zviwire pasi semvura yakaoma mushure mekuburitsa.

Zvinetso zvinoramba zvichidzokorora

Mabhendi ejeli ane ruvara rusina kusimba anowanzoreva kuti huwandu hwezvinhu hushoma kuti huonekwe nedhayi rinoonekwa neruvara, kunyanya pazvinhu zvipfupi zvetambo imwe kana zveRNA. Isa zvakawanda, shandisa dhayi idzva, wedzera nguva yekuisa ruvara usati wawedzera kusimba kwemusanganiswa, uye mira kubvisa ruvara nguva ichiri pfupi. Jeli rakazara bhuruu risina musiyano unooneka rinowanzoreva kuisa ruvara zvakanyanya kana kuchinja mvura kashoma. Mabhendi ePAGE ari kupera ruvara ndiwo maitiro emakemikari anotarisirwa, kwete kukundikana kwedhayi. Tora mufananidzo nokukurumidza. Ruvara rwemashure rusina kuenzana pamembrane runoratidza mapete, mafuro, zvisaririra zveminwe, kuzunza kusina kuenzana, kana kutamisa kwakaitika kune dzimwe nzvimbo chete. Usadudzira izvi sekusiyana kwehuwandu hwakaiswa pasina bvunzo yekudzora yehybridization. Patani yeribosomal isina kusimba uye yakapararira inogona kureva kuparara, asi inogona kurevawo kutamisa kusina kukwana, RNA yose iri shoma (migero yakajairika inotakura ma micrograms akati wandei kusvika kumakumi), kana kubvisa ruvara zvakanyanya. Dhayi rinoratidza ribosomal RNA yakawanda. Hariratidzi transcript yakati. Basa iroro ndereprobe.

Kuchengeteka nekubata pasina kuwedzeredza

Methylene blue iri nyore kubata pane ethidium bromide, asi haisi isina njodzi. Gwaro razvino reMilliporeSigma remashoko ekuchengeteka rinoronga dhayi seCategory 4 yechepfu inokurumidza kukuvadza kana yamedzwa, ichikuvadza kana ikamedzwa, uye rinoda kuraswa nenzira yakatenderwa yemarara maererano neSDS yemethylene blue yeSigma-Aldrich. Ethidium bromide ine yambiro dzakasimba zvikuru dzekukuvadza zvinhu zvemajini, kusanganisira kufungidzirwa kwekukanganisa masero ekubereka muSDS ye ethidium bromide yeSigma-Aldrich, uye majeli nemisanganiswa yayo zvinowanzoparadzaniswa semarara emakemikari ane ngozi sezvakanyorwa mugwaro reThermo Fisher remadhayi enucleic acid. Tevedzera SDS nemitemo yesangano pazvose zviri zviviri. Chengeta dhayi rakaoma rakavharwa zvakasimba uye rakaoma, shandisazve dhayi riri kushandiswa chete sezvinobvumirwa nemaitiro, chengeta mamembrane akaiswa ruvara akaoma pakati pemapepa ekusefa, uye tora mifananidzo yemajeli mabhendi asati apararira.

Rangarira mutsara wekushanda murabhoritari. Majeli anoedza kuoneka kwezvinhu zvakaiswa muhuwandu hwakawanda pachiedza chichena. Mamembrane anoedza kutamisa nehuwandu hwakaiswa probe isati yaita basa rekuona zvinhu zvishoma. Dhayi rimwe chete rinoshanda pazvose zviri zviviri, nekugona kwakaderera kuona zvinhu zvishoma, kusunga kunogona kudzoserwa shure, uye kurongeka kwematanho kunofanira kunyatsotevedzwa.