Methylene blue podaya DNA ndi RNA: kugwiritsa ntchito m'majelu ndi pa nembanemba

Wophunzira amachita electrophoresis pa jelu ya agarose, amaidaya ndi methylene blue, ndipo saona chilichonse pamene ankayembekezera mzere wowala. Pamalo ogwirira ntchito oyandikana nawo, wofufuza amene wamaliza digiri ya udokotala amadaya nembanemba ya Northern ndi botolo lomwelo la dayi yabuluu ndipo amaona mizere yomveka bwino ya RNA ya ribosome m'mphindi zitatu. Wophunzirayo amaganiza kuti kuyesa kwa jelu kwalephera. Koma sikunalephere. Ntchito ziwirizi zinali zosiyana, ndipo dayi yomweyo imayankha mafunso awo ndi mphamvu yosiyana yozindikira, mtundu wosiyana wakumbuyo, ndi malamulo osiyana a zomwe ziyenera kuchitika pambuyo pake.

Kudaya DNA ndi methylene blue ndi kudaya RNA ndi methylene blue zimagwiritsa ntchito khemistri yofanana, koma zolinga zake n'zosiyana. Mu jelu, dayiyo imasonyeza kuchuluka kwa nucleic acid komwe kuli mkati mwa jelu yokhala ndi timabowo. Pa nembanemba, imasonyeza ngati kusamutsa kunayenda bwino komanso ngati zitsanzo zinaikidwa mofanana m'misewu ya jelu. Kusiyana kumeneku kukamveka bwino, njira zogwirira ntchito, malire ozindikira, ndi malire a kugwirizana ndi njira zina zimamvekanso bwino.

Yankho lachidule

Methylene blue ndi dayi ya mamolekyulu athyathyathya yokhala ndi chaji yabwino. Imamangirira ku ma nucleic acid popanda kupanga zomangira za covalent ndipo ingachotsedwenso. Izi zimachitika mwa kuphatikiza kukopana kwa chaji ndi phosphate, kulowerera pakati pa mapeya a mabesi, ndi kumangirira mu ngalande yaing'ono ya DNA. Kutha kuchotsa dayiyo ndiko kofunika. Mizere ingadayidwe, kuonedwa ndi kuwala kooneka, kujambulidwa, kenako dayiyo kuchotsedwa kapena DNA kuyeretsedwa kuti igwiritsidwe ntchito popanga makope a DNA, pa hybridization, kapena poyesanso ndi probe.

Podaya DNA ya zingwe ziwiri mu jelu ya agarose ndi methylene blue, njira yachangu yomwe imagwiritsidwa ntchito kawirikawiri ndi 0.02 percent w/v (200 micrograms per mL) m'madzi, kudaya kwa pafupifupi 15 minutes kenako kuchotsa dayi ndi madzi kwa pafupifupi 15 minutes, ndi kuyang'ana pa bokosi la kuwala koyera. Pa nembanemba, njira yakale ya Northern blot imagwiritsa ntchito dayi ya 0.02 mpaka 0.04 percent mu sodium acetate pafupi ndi pH 5.2, kudaya kwa mphindi zingapo pambuyo posamutsa ndi kukhazikitsa zitsanzo kuti zisachoke, kenako kutsuka ndi madzi ndi kuchotsa dayi yonse musanachite hybridization. Mphamvu yozindikira ndi yochepa: makumi mpaka mazana a nanograms pa mzere uliwonse wa jelu pamikhalidwe yabwino, ndipo malangizo osamala amanena 0.5 mpaka 1 microgram. Kuchuluka kumeneko ndi kwakukulu ndi magawo angapo a khumi poyerekeza ndi komwe madayi a ethidium bromide ndi SYBR angazindikire. Pa nembanemba palibe malire amodzi othandiza ozindikira transcript inayake, chifukwa dayiyo imasonyeza makamaka RNA yochuluka ya ribosome ndi zinthu zonse zomwe zinaikidwa. Musalowe m'malo mwa dayi ya njira iliyonse imeneyi ndi yankho la chowonjezera chakudya chogulitsidwa m'masitolo. Njira zotsimikiziridwa zimafuna dayi ya mulingo woyenera wa kusanthula mu labotale, ma buffer okhala ndi kapangidwe kodziwika, ndi kugwira ntchito popanda RNase pamene RNA ikukhudzidwa.

Mmene kumangirira kumachitikira, ndi pamene kungasocheretse

Chitsanzo chothandiza chimakhala ndi mbali zitatu. Choyamba, kapangidwe ka mphete za mamolekyulu kokhala ndi chaji yabwino kamakopeka ndi msana wa phosphate wokhala ndi chaji yoipa. Kafukufuku wa spectroscopy amapeza kuti kukopana kwa chaji kumeneku n'kumene kumalamulira mchere ukakhala wochepa, pamene zitsanzo za pakompyuta ndi kafukufuku wa mayendedwe a mamolekyulu zimasonyeza kuti kulowerera pakati pa mabesi ndi kumangirira mu ngalande yaing'ono ndi njira zokhazikika pa DNA ya zingwe ziwiri, monga momwe zafotokozedwera mu kafukufuku wa spectroscopy wokhudza kumangirira kwa methylene blue ku DNA. Chachiwiri, pali kukonda pang'ono malo a GC. Zitsanzo za pakompyuta zimakonda masitepe a CpG ndi GpC, ndipo mayesero apeza kumangirira kwambiri ku zingwe ziwiri zokhala ndi GC yambiri mu kafukufuku wa malo amene methylene blue imakonda kulowerera, koma imamangiriranso ku zingwe ziwiri za AT. Kudaya kwa nthawi zonse sikumasankha ndondomeko inayake ya mabesi. Chachitatu, DNA ya chingwe chimodzi ndi RNA zimamangirira makamaka chifukwa cha kukopana kwa chaji ndi kusanjikana ndi mabesi opezeka mosavuta ndi madera a zingwe ziwiri. N'chifukwa chake mphamvu ya kudaya imasiyanasiyana malinga ndi kutalika, ndondomeko ya mabesi, ndi kapangidwe kopindika ka chingwecho.

Pali zotsatira ziwiri. Popeza kumangirira sikukhala kwa covalent, dayi imachoka potsuka. Mizere ya polyacrylamide imafota msanga pokhapokha ngati yakhazikitsidwa mwadala kuti isafote. Pali njira yapadera yokhazikitsira mtundu kosatha chifukwa kudaya kwanthawi zonse n'kwakanthawi, monga momwe Soto ndi Draper anafotokozera pa majelu a PAGE. Popeza dayiyo imayambitsa kusintha kwa khemistri ikalandira kuwala, kuunikira DNA yodayidwa ndi kuwala kwamphamvu kwa nthawi yaitali pamene oxygen ilipo kungapangitse oxidation ya guanine ndipo pamapeto pake kudula zingwe pang'ono. Njirayi yafotokozedwa mu kafukufuku wa oxidation ya guanine yoyambitsidwa ndi methylene blue ikalandira kuwala. Kuyang'ana mwachidule ndi kuwala koyera kumapewa kuwonongeka kodziwika kwa UV komwe kumachitika m'njira zakale za ethidium bromide, ndipo ubwino umenewo ndi weniweni pochotsa DNA kuti igwiritsidwenso ntchito. Kuwala kooneka kumawononga pang'ono, koma sikuli kopanda chiopsezo. Chepetsani nthawi younikira ngati DNA idzagwiritsidwa ntchito popanga makope. Kuti mudziwe zambiri za khemistri ya kuwala yomwe imachititsa chenjezo limeneli, onani njira ziwiri za khemistri ya methylene blue ikalandira kuwala. Kuti mudziwe mitundu iwiri ya redox ndi mikhalidwe ya chaji yomwe imafotokoza chifukwa chake mtundu wa oxidized umamangirira koma wa reduced sumamangirira, onani khemistri ya kapangidwe ndi mikhalidwe ya methylene blue.

Ntchito yoyamba: kudaya mkati mwa majelu

Kudaya jelu kumayankha funso limodzi: kodi mu mzerewu muli nucleic acid yokwanira kuti ioneke ndi maso? DNA ya zingwe ziwiri mu agarose ndiyo yafufuzidwa bwino kwambiri. Njira yoyambirira ya Flores ndi anzake yodaya jelu ndi methylene blue inatsimikizira njira yachangu ya 0.02 percent pa agarose ya 1 percent ndipo inasonyeza kuti DNA ingachotsedwe bwino ndi kugwiritsidwa ntchito popanga makope pambuyo pake. Njira yomweyo ya wogulitsa imapezeka pa tsamba la methylene blue la IBI Scientific, lomwe limalemba limodzi njira ya nembanemba ya 0.04 percent ndi njira yachangu ya jelu.

Njira yogwirira ntchito:

1. Chitani electrophoresis pa jelu monga mwa nthawi zonse. Dayani pambuyo pa electrophoresis, osati pamene ikuchitika, pokhapokha ngati kusamutsa kapena kulekanitsa zitsanzo kwatsimikiziridwa pa njira yosinthidwa.
2. Mizamitsani mu methylene blue ya 0.02 percent w/v m'madzi osungunulidwa ndi nthunzi, ndikuigwedeza pang'onopang'ono, kwa pafupifupi 15 minutes.
3. Chotsani dayi posintha madzi kangapo, kwa pafupifupi 15 minutes zonse pamodzi, mpaka mtundu wakumbuyo wa jelu uchepe ndipo mizere ioneke bwino.
4. Jambulani chithunzi msanga pa chipangizo chounikira ndi kuwala koyera.

Agarose ndi polyacrylamide zimachita mosiyana. Agarose imasunga dayi mkati mwa jelu, choncho kuchotsa dayi ndiko kumatenga nthawi. Majelu opyapyala a polyacrylamide amadayika msanga koma amatayanso dayi msanga. Izi zimathandiza podula zidutswa kuti zigwiritsidwenso ntchito, koma zimapangitsa kukhala kovuta kusunga umboni wooneka kwa nthawi yaitali. Ma oligonucleotide opangidwa mu labotale omwe ali mu polyacrylamide angapezeke pogwiritsa ntchito njira yomweyo ya 0.02 percent kwa 20 mpaka 30 minutes ndi kutsuka mobwerezabwereza ndi madzi, potsatira njira zokhazikika za PAGE za mtundu wa Current Protocols. Zanenedwanso za njira zochepetsedwa kwambiri mpaka pafupi ndi 0.0002 percent mu TBE yochepetsedwa, zogwiritsidwa ntchito pa ma oligo oti adzagwiritsidwenso ntchito kwa maola angapo, koma umboni wotsimikizira njira zimenezi ndi wochepa.

Kusiyana pakati pa DNA ndi RNA n'kofunika. DNA ya zingwe ziwiri imamangirira mwamphamvu kwambiri m'njira yokhazikika. RNA ndi DNA ya chingwe chimodzi zimadayika, koma mofooka komanso m'njira yosavuta kusiyanasiyana. Pa majelu a RNA a formaldehyde agarose, malabotale ambiri samadaya ndi buluu mkati mwa jelu. Amasamutsa RNA kupita pa nembanemba kaye, kenako n'kuidaya pamenepo. Kusintha kumeneko kumachotsa chizindikiro chofooka cha jelu n'kupeza chizindikiro chomveka bwino pa nembanemba komanso umboni wa kuchuluka kwa zitsanzo zoikidwa.

Ntchito yachiwiri: kudaya pa nembanemba

Kudaya nembanemba kumayankha funso lina: kodi kusamutsa kunayenda bwino, ndipo misewu ya zitsanzo ingafanane? Njira yakale ndi dayi ya Northern blot ya Herrin ndi Schmidt yomwe ingachotsedwenso. Poyambirira inkagwiritsa ntchito dayi ya 0.04 percent mu sodium acetate ya 50 mM pa pH 5.0 kwa 2 mpaka 3 minutes, kenako kutsuka ndi madzi. Dayiyo inkachotsedwa ndi SDS yochepetsedwa mu SSPE musanachite hybridization.

Njira yamakono yomwe imagwiritsidwa ntchito kwambiri imayenda motere:

1. Samutsani RNA kapena DNA yomwe zingwe zake zalekanitsidwa kupita pa nylon kapena nitrocellulose, kenako ikhazikitseni mwa kulumikiza ndi UV kapena kutenthetsa kuti chitsanzocho chisatayike podaya.
2. Izi si zokakamiza koma zimachitika kawirikawiri: nyowetsani nembanembayo mu acetic acid ya 5 percent kwa pafupifupi 15 minutes.
3. Dayani kwa 5 mpaka 10 minutes mu methylene blue ya 0.02 mpaka 0.04 percent mu sodium acetate ya 0.3 mpaka 0.5 M pafupi ndi pH 5.2.
4. Tsukani ndi kuchotsa dayi m'madzi kwa 5 mpaka 15 minutes, lembani umboni kapena jambulani, kenako chotsani dayi yotsalayo musanachite hybridization.

Nylon yokhala ndi chaji yabwino ndiyo imagwiritsidwa ntchito kwambiri pa Northern. Nitrocellulose imagwiranso ntchito ndi njira yomweyo ya acetic acid ndi acetate. Chithunzicho chimagwiritsidwa ntchito kutsimikizira kusamutsa, kuyerekezera mizere ya 28S ndi 18S (kapena 23S ndi 16S pa RNA ya mabakiteriya), kulemba malo a zizindikiro za kukula ndi zitsanzo zoyezera, ndi kusunga mbiri yoyerekeza kuchuluka kwa RNA yonse kuti zitsanzo zifanane. Njira zofalitsidwa za Northern zimayesa kuchuluka kwa mizere yodayidwa ya RNA ya ribosome poyang'ana kuchuluka kwa zitsanzo zoikidwa, kuphatikizapo kafukufuku wa sayansi ya mkaka amene anagwiritsa ntchito mizere ya ribosome yodayidwa ndi methylene blue poyerekeza kuchuluka kwa zitsanzo. Nembanemba za Southern zingaonedwenso mwa njira yomweyo, koma dayiyo yakhazikika kwambiri ngati njira yoyang'anira kuchuluka kwa zitsanzo ndi kusamutsa pa Northern. Komanso, zingwe za DNA ya Southern zimalekanitsidwa musanasamutse, choncho manambala a mphamvu yozindikira mu jelu sagwira ntchito mofanana pamenepo.

Malire ozindikira ndi mtundu wakumbuyo: pamene buluu imalephera kupikisana

Kuyerekezera pansipa ndi kwa pafupifupi mwadala. Mmene dayi imagwirira ntchito zimadalira kuchuluka kwa zinthu zopanga jelu, m'lifupi mwa mzere, zida zojambulira kuwala, ndi kusamala pochotsa dayi. Tengani nambala iliyonse ngati mulingo wa kuchuluka, osati monga muyezo wotsimikizika.

Kuchepetsa mtundu wakumbuyo ndiko kumatsimikizira ngati chizindikiro chochepacho chingagwiritsidwe ntchito. Dayi yosamangirira imakhala ndi mtundu wamphamvu kwambiri, choncho kusiyanitsa mizere ndi kumbuyo kumatheka pochotsa dayi yosamangirira popanda kuchotsa yomangirira. Dayi yochuluka kwambiri si kawirikawiri imene imawonjezera mphamvu yozindikira. Makamaka imangowonjezera nthawi yochotsa dayi. Yesani zitsanzo za kuchuluka kochepetsedwa motsatizana pa jelu, nembanemba, ndi kamera zomwe mugwiritse ntchito, ndipo gwiritsani ntchito kuchuluka kokhako kumene muyeso wonse wa kulimba kwa mtundu ukusintha molingana ndi kuchuluka kwa chitsanzo choikidwa. Methylene blue imathandiza poyerekeza kuchuluka kwa zitsanzo mwachiyerekezo. Si njira yoyezera molondola pa kuchuluka kosiyanasiyana kwambiri, chifukwa mphamvu ya nembanemba yosunga zinthu, kuyenda bwino kwa kusamutsa, kufika pa malire a kudzaza, ndi kuchotsa dayi zimasiyanasiyana pakati pa misewu ya zitsanzo.

Malire a kugwirizana: zomwe zingagwiritsidwe ntchito pambuyo podaya

Kupanga makope a DNA pambuyo podaya ndi buluu kwasonyezedwa kuti kumatheka. Flores ndi anzake anadula mizere yodayidwa ya agarose, anachotsamo DNA, kenako anailumikiza ndi kuiika m'maselo. Iwo ananena kuti kupanga makope kunayenda bwino kuposa pambuyo pogwiritsa ntchito ethidium bromide ndi UV. Chifukwa choyembekezeredwa ndi kuchepa kwa kuwonongeka kochitika chifukwa cha kuwala, osati ubwino wochokera ku dayiyo yokha. Chepetsani nthawi yoonetsa DNA ku kuwala koyera, ndipo yeretsani DNA yochotsedwayo ndi njira yanthawi zonse yoyeretsera DNA yochokera mu jelu.

Umboni wokhudza PCR ndi kupeza ndondomeko ya mabesi ndi wochepa. Dayi yotsala imamangirira ku ma nucleic acid, choncho chotsani dayi ndi kuyeretsa musanachite ntchito zogwiritsa ntchito ma enzyme. DNA yoyeretsedwa bwino yochokera mu jelu yabuluu ikuyembekezeredwa kugwira ntchito, koma kuchuluka kwa dayi yotsala komwe ma polymerase ndi njira zosiyanasiyana zopezera ndondomeko ya mabesi zingapirire sikunayezedwe bwino. Musangoganiza kuti sizingakhudze ntchitoyo.

Hybridization ya blot imagwirizana ndi kudaya ngati dongosolo litsatidwa mosamalitsa: kusamutsa, kukhazikitsa kuti zitsanzo zisachoke, kudaya, kusunga umboni, kuchotsa dayi, ndi hybridization. Limenelo ndilo dongosolo la Herrin ndi Schmidt. Kudaya musanasamutse, kapena kusachotsa dayi, ndi njira ina ndipo iyenera kutsimikiziridwa payokha. Dayi ikachotsedwa, kuchotsa probe yakale ndi kuyesanso ndi probe yatsopano kumachitika monga mwa nthawi zonse. Kuti mudziwe za kudaya maselo ndi gulu lomwelo la madayi ndiponso chifukwa chake mfundo za zitsanzo zokhazikitsidwa pa slide sizigwira ntchito mofanana pa maselo amoyo, onani atlas ya kudaya poyang'ana ndi maikulosikopu.

Kudula mizere kumagwira ntchito pa agarose yogwiritsidwa ntchito pokonza DNA kuti idzagwiritsidwenso ntchito komanso pa ma oligo a PAGE opezeka ndi buluu, ngati njira yokhazikitsira mtundu kosatha siyagwiritsidwa ntchito. Kuchotsa zitsanzo mu jelu ndi magetsi pambuyo pochotsa dayi n'kotheka malinga ndi khemistri ya chinthu chomangirira chomwe chingachotsedwenso, koma deta ya mayesero olamulidwa yokhudza kuchuluka kwa zitsanzo zomwe zimapezeka pogwiritsa ntchito methylene blue ndi yochepa. Chotsani mchere kapena chititsani kuti zitsanzo zituluke m'yankho pambuyo pozichotsa mu jelu.

Zolakwika zomwe zimabwerezabwereza

Mizere yofooka ya jelu nthawi zambiri imatanthauza kuti kuchuluka kwa zinthu n'kochepa kwambiri kuti dayi yooneka ndi mtundu izizindikire, makamaka pa zitsanzo zazifupi za chingwe chimodzi kapena RNA. Ikani chitsanzo chochuluka, gwiritsani ntchito dayi yatsopano, onjezerani nthawi yodaya musanawonjezere kuchuluka kwa dayi, ndipo siyani kuchotsa dayi msanga. Jelu yabuluu yonse yopanda kusiyana kwa mizere ndi kumbuyo nthawi zambiri imatanthauza kudaya mopitirira muyeso kapena kusintha madzi kaŵerengero kochepa. Kufota kwa mizere ya PAGE ndi zotsatira zoyembekezeredwa za khemistri, osati kulephera kwa dayi. Jambulani msanga. Mtundu wakumbuyo wosafanana pa nembanemba umasonyeza makwinya, thovu, zidindo za zala, kugwedeza kosafanana, kapena kusamutsa kosafanana m'malo osiyanasiyana. Musamasulire izi kuti ndi kuchuluka kosafanana kwa zitsanzo popanda kuyesa kolamulira kwa hybridization. Mizere yofooka ya ribosome yofalikira ingatanthauze kuwonongeka kwa RNA, komanso kusamutsa kosakwanira, RNA yonse yochepa kwambiri (misewu yanthawi zonse imakhala ndi micrograms angapo mpaka makumi), kapena kuchotsa dayi mopitirira muyeso. Dayiyo imasonyeza RNA yochuluka ya ribosome. Sisonyeza transcript inayake. Ntchito imeneyo ndi ya probe.

Chitetezo ndi kasamalidwe popanda kunena zopitirira umboni

Methylene blue ndi yosavuta kugwiritsa ntchito kuposa ethidium bromide, koma si yopanda chiopsezo. Pepala laposachedwapa la MilliporeSigma la chidziwitso cha chitetezo limaika dayiyo mu Category 4 ya poizoni wadzidzidzi akamezedwa, kuti ndi yovulaza ikamezedwa, ndipo zinyalala zake ziyenera kuchotsedwa kudzera m'njira yovomerezeka malinga ndi SDS ya methylene blue ya Sigma-Aldrich. Ethidium bromide ili ndi machenjezo amphamvu kwambiri okhudza kuwononga zinthu za chibadwa, kuphatikizapo kukayikiridwa kuti imakhudza maselo oberekera mu SDS ya ethidium bromide ya Sigma-Aldrich. Majelu ndi mayankho ake nthawi zambiri amasiyanitsidwa ngati zinyalala za mankhwala oopsa, monga momwe zalembedwera mu buku la Thermo Fisher lofotokoza madayi a nucleic acid. Tsatirani SDS ndi malamulo a bungwe lanu pa zonsezi. Sungani dayi youma ili yotsekedwa mwamphamvu komanso youma, gwiritsaninso ntchito dayi yokonzedwa pokhapokha ngati njira yogwirira ntchito ikulola, sungani nembanemba zodayidwa zouma pakati pa mapepala osefera, ndipo jambulani majelu mizere isanabalalike.

Kumbukirani chiganizo cha pa tebulo la labotale. Majelu amayesa kuoneka kwa zitsanzo zambiri pansi pa kuwala koyera. Nembanemba zimayesa kusamutsa ndi kuchuluka kwa zitsanzo probe isanachite ntchito yozindikira zinthu zochepa kwambiri. Dayi imodzi imagwira ntchito zonse ziwiri, ndi mphamvu yochepa yozindikira, kumangirira komwe kungathetsedwe, ndi dongosolo la ntchito lomwe liyenera kutsatidwa mosamalitsa.