I-methylene blue yokudaya i-DNA ne-RNA: ukusetyenziswa kwiijeli nakwiimembrane

Umfundi uqhuba ijeli ye-agarose, ayidaye nge-methylene blue, aze angaboni nto apho bekulindeleke ibhendi eqaqambileyo. Kwindawo yokusebenzela esecaleni, umphandi ogqibe isidanga sobugqirha udaya imembrane ye-Northern ngedayi ekwibhotile enye eluhlaza okwesibhakabhaka, aze afumane iibhendi zeribosome ezicacileyo kwimizuzu emithathu. Umfundi ugqiba kwelokuba ijeli ayisebenzanga. Ijeli ayikhange isilele. Le misebenzi mibini ibahlukile, yaye idayi enye iyiphendula ngobuntununtunu obahlukileyo, ngombala ongemva owahlukileyo, nangemithetho eyahlukileyo yamanyathelo alandelayo.

Ukudaya i-DNA nge-methylene blue nokudaya i-RNA nge-methylene blue kusebenzisa ikhemistri efanayo kodwa kuneenjongo ezahlukileyo. Kwijeli, idayi ibonisa ubungakanani be-asidi yenukliyiki ekhoyo ngaphakathi kwisakhiwo esinemingxuma. Kwimembrane, ibonisa ukuba ukudluliselwa kuphumelele na nokuba iindlela zifakwe izixa ezilinganayo na. Xa lo mahluko ucacile, iiprothokholi, imida yokufumanisa, nemida yokuhambelana ziyavakala.

Impendulo emfutshane

I-methylene blue yidayi enesakhiwo esisicaba nentlawulo yombane elungileyo, ebophelela kwii-asidi zenukliyiki ngaphandle kweebhondi zekhovalenti nangendlela ebuyisekayo. Isebenzisa indibaniselwano yokutsaleleka kwe-electrostatic kwi-phosphate, ukungena phakathi kwezibini zeebheyisi, nokubophelela kumjelo omncinane. Oko kubuyiseka yeyona nto ibalulekileyo. Iibhendi zinokudaywa zize zijongwe ngokukhanya okubonakalayo, zibhalwe phantsi, emva koko kususwe idayi okanye zicocwe ukuze zisetyenziselwe ukuklona, ihayibridayizeshini, okanye ukuphinda zivavanywe ngeprobe.

Ekudayweni kwe-DNA enemisonto emibini kwiijeli ze-agarose nge-methylene blue, umxube oqhelekileyo okhawulezayo yi-0.02 percent w/v (200 micrograms per mL) emanzini, malunga nemizuzu eyi-15 yokudaya kunye nemizuzu emalunga ne-15 yokususa idayi ngamanzi, kuze kujongwe kwibhokisi yokukhanya okumhlophe. Kwiimembrane, umxube wakudala we-Northern blot usebenzisa idayi eyi-0.02 ukuya ku-0.04 percent kwi-sodium acetate kufutshane ne-pH 5.2, imizuzu eliqela yokudaya emva kokudluliselwa nokumiliselwa, kulandele ukuhlanjwa ngamanzi nokususwa ngokupheleleyo kwedayi ngaphambi kwehayibridayizeshini. Ubuntununtunu buphakathi: ukusuka kumashumi ukuya kumakhulu ee-nanograms kwibhendi nganye yejeli phantsi kweemeko ezifanelekileyo, yaye izikhokelo ezithatha uqikelelo olulumkileyo zidwelisa i-0.5 ukuya kwi-1 microgram. Ezo zixa zingaphezulu ngemiyalelo emininzi yobukhulu kunezo zifunyaniswa nge-ethidium bromide needayi ze-SYBR. Kwiimembrane akukho mda omnye oluncedo wokufumanisa umbhalo othile we-RNA, kuba idayi ibonisa ikakhulu i-RNA yeribosome eninzi nesixa sonke esifakiweyo. Musa ukufaka isinyibilikiso sesongezelelo esithengiselwa uluntu endaweni yayo kuwo nawuphi na umxube. Iindlela eziqinisekisiweyo zicingela ukusetyenziswa kwedayi ekumgangatho wohlalutyo, izinyibilikiso ezigcina i-pH ezinokwakheka okuchaziweyo, nokuphathwa ngaphandle kwe-RNase xa kusetyenziswa i-RNA.

Indlela ukubophelela okusebenza ngayo, nalapho kunokulahlekisa khona

Imodeli eluncedo inamacandelo amathathu. Okokuqala, isakhiwo samakhonkco esinentlawulo yombane elungileyo sitsaleleka kumqolo we-phosphate onentlawulo engalunganga. Uphando lwe-spectroscopy lufumanisa ukuba eli galelo le-electrostatic lelona lilawulayo xa ityuwa iphantsi, ngoxa iimodeli ne-dynamics yeemolekyuli zibonisa ukuba ukungena phakathi kwezibini zeebheyisi nokubophelela kumjelo omncinane ziindlela ezizinzileyo kwi-DNA enemisonto emibini, njengoko kushwankathelwe kuphononongo lwe-spectroscopy lokubophelela kwe-methylene blue kwi-DNA. Okwesibini, kukho ukhetho oluthile lwe-GC: iimodeli zikhetha amanyathelo e-CpG ne-GpC, yaye iimvavanyo zibonisa ukubophelela okukhethayo kwimicu emibini etyebileyo nge-GC kuphando lokhetho lwe-methylene blue xa ingena phakathi kwezibini zeebheyisi, kodwa nemicu emibini ye-AT iyabophelela. Ukudaya okuqhelekileyo akukhethi ulandelelwano oluthile. Okwesithathu, i-DNA enomsonto omnye ne-RNA zibophelela ikakhulu nge-electrostatic nangokupakishana neebheyisi ezifikelelekayo nemimandla enemisonto emibini. Yiyo loo nto amandla okudaya exhomekeke kubude, kulandelelwano, nakwisakhiwo sesibini.

Kulandela iziphumo ezimbini. Kuba ukubophelela akusebenzisi iibhondi zekhovalenti, idayi iyaphuma xa ihlanjwa. Iibhendi ze-polyacrylamide ziphelelwa ngumbala ngokukhawuleza ngaphandle kokuba zimiliselwe ngabom, yaye kukho indlela ekhethekileyo yokumilisela ngokusisigxina kanye kuba ukudaya okuqhelekileyo kokwexeshana, njengoko kuchazwa nguSoto noDraper kwiijeli ze-PAGE. Kuba idayi yinto evuselelwa kukukhanya, ukukhanyiselwa ixesha elide ngokukhanya okuqaqambileyo kwe-DNA edayiweyo xa kukho ioksijini kunokwenza i-guanine ibe ne-oxidation kuze ekugqibeleni kubangele ukusikeka kwemisonto. Le ndlela ichazwe kuphando lwe-oxidation ye-guanine evuselelwa yi-methylene blue. Ukujonga ngokufutshane ngokukhanya okumhlophe kuthintela umonakalo owaziwayo we-UV kwiinkqubo zakudala ze-ethidium bromide, yaye olu ncedo luyinyani xa kufunyanwa i-DNA ukuze isetyenziswe kamva. Ukukhanya okubonakalayo kuthambile ngakumbi, kodwa akukhuselekanga ngokupheleleyo. Gcina ixesha lokukhanyisela lifutshane xa i-DNA iza kuklonwa. Ukufumana ingcaciso ebanzi ngekhemistri yokukhanya exhasa eso silumkiso, jonga iindlela ezimbini zekhemistri yokukhanya ye-methylene blue. Ukufumana ingcaciso ngesibini se-redox neemeko zentlawulo ezichaza ukuba kutheni imo ene-oxidation ibophelela kodwa imo encitshisiweyo ingabopheleli, jonga ikhemistri yesakhiwo neempawu ze-methylene blue.

Umsebenzi wokuqala: ukudaya ngaphakathi kwiijeli

Ukudaya ijeli kuphendula umbuzo omnye: ingaba ikhona i-asidi yenukliyiki eyaneleyo kule bhendi ukuze ibonwe ngeliso? I-DNA enemisonto emibini kwi-agarose yeyona meko ifundwe yaza yachazwa kakuhle. Indlela yokuqala yokudaya ijeli nge-methylene blue kaFlores noogxa bakhe yaqinisekisa iprothokholi ekhawulezayo ye-0.02 percent kwi-agarose eyi-1 percent, yaza yabonisa ukufunyanwa nokuklonwa okuyimpumelelo emva koko. Iprothokholi efanayo yomthengisi ivela kwiphepha lemveliso ye-methylene blue le-IBI Scientific, elidwelisa umxube weemembrane we-0.04 percent kunye nendlela ekhawulezayo yeejeli ecaleni komnye.

Inkqubo yokusebenza:

1. Qhuba ijeli ngendlela eqhelekileyo. Daya emva kwe-electrophoresis, kungekhona ngexesha layo, ngaphandle kokuba ukudluliselwa okanye amandla okwahlula aqinisekisiwe kwindlela eguquliweyo.
2. Yintywilisele kwi-methylene blue eyi-0.02 percent w/v emanzini adistiliweyo, uyishukumise ngobunono, malunga nemizuzu eyi-15.
3. Susa idayi ngokutshintsha amanzi izihlandlo ezininzi, malunga nemizuzu eyi-15 iyonke, de umbala ongemva wesakhiwo sejeli unciphise zize iibhendi zicace.
4. Yifote ngokukhawuleza kwisixhobo sokukhanyisela ngokukhanya okumhlophe.

I-agarose ne-polyacrylamide ziziphatha ngokwahlukileyo. I-agarose igcina idayi kwisakhiwo sayo, ngoko ukususa idayi linyathelo elicothayo. Iijeli ezibhityileyo ze-polyacrylamide zidayeka ngokukhawuleza kodwa ziphulukana nedayi ngokukhawuleza, nto leyo enceda xa kusikwa iibhendi ukuze zisetyenziswe kamva kodwa ingalungelanga ukugcina iirekhodi. Ii-oligonucleotide ezenziwe ngokwenziwa kwi-polyacrylamide zinokufunyanwa ngomxube ofanayo we-0.02 percent kwimizuzu eyi-20 ukuya ku-30 ngokuzihlamba ngamanzi ngokuphindaphindiweyo, kulandelwa iindlela ze-PAGE ezimiselweyo zohlobo lwe-Current Protocols. Kuye kwaxelwa iindlela ezixutywe kakhulu, ezikufutshane ne-0.0002 percent kwi-TBE exutyiweyo, zokulungiselela ii-oligo kwiiyure eziliqela, kodwa ubungqina bazo bubuthathaka ngakumbi.

Umahluko phakathi kwe-DNA ne-RNA ubalulekile. I-DNA enemisonto emibini ibonelela ngokubophelela okucwangcisekileyo okunamandla kakhulu. I-RNA ne-DNA enomsonto omnye ziyadayeka, kodwa ngobuthathaka ngakumbi nangendlela engaqikeleleki lula. Kwiijeli ze-RNA ze-agarose ezine-formaldehyde, iilabhoratri ezininzi azidayi ngeblue ngaphakathi kwejeli; ziqala ngokudlulisela i-RNA kwimembrane, zize ziyidaye apho. Oku kutshintsha uphawu olubuthathaka lwejeli kube luphawu olucacileyo ngakumbi lwemembrane, kunye nerekhodi yezixa ezifakiweyo.

Umsebenzi wesibini: ukudaya kwiimembrane

Ukudaya imembrane kuphendula umbuzo owahlukileyo: ingaba ukudluliselwa kusebenzile, yaye iindlela ziyafaniswa? Ukusetyenziswa kwakudala yidayi ebuyisekayo ye-Northern blot kaHerrin noSchmidt, eyayiqala nge-0.04 percent yedayi kwi-50 mM sodium acetate kwi-pH 5.0 imizuzu eyi-2 ukuya ku-3, kulandelwe ukuhlanjwa ngamanzi. Idayi yayisuswa nge-SDS exutyiweyo kwi-SSPE ngaphambi kwehayibridayizeshini.

Indlela yale mihla esetyenziswa ngokubanzi ilandela la manyathelo:

1. Dlulisela i-RNA okanye i-DNA enesakhiwo esiqhawuliweyo kwinayiloni okanye kwi-nitrocellulose, uze uyimilisele ngokuyidibanisa nge-UV okanye ngokuyibhaka ukuze isampulu ingalahleki ngexesha lokudaya.
2. Linyathelo elinganyanzelekanga kodwa eliqhelekileyo: manzisa imembrane kwi-5 percent acetic acid malunga nemizuzu eyi-15.
3. Daya imizuzu eyi-5 ukuya ku-10 kwi-methylene blue eyi-0.02 ukuya ku-0.04 percent kwi-0.3 ukuya ku-0.5 M sodium acetate kufutshane ne-pH 5.2.
4. Hlamba uze ususe idayi ngamanzi imizuzu eyi-5 ukuya ku-15, bhala phantsi iziphumo, emva koko ususe idayi eseleyo ngaphambi kwehayibridayizeshini.

Inayiloni enentlawulo yombane elungileyo yeyona isetyenziswa kakhulu kumsebenzi we-Northern. I-nitrocellulose nayo iyasebenza ngenkqubo efanayo ye-acetic acid kunye ne-acetate. Umfanekiso usetyenziselwa ukuqinisekisa ukudluliselwa, ukuthelekisa iibhendi ze-28S ne-18S (okanye i-23S ne-16S kwi-RNA yeebhaktheriya), ukuphawula iileli nemigangatho, nokugcina irekhodi eliqikelelweyo lokulungelelanisa ngokwesixa sonke se-RNA. Iindlela ze-Northern ezipapashiweyo zilinganisa iibhendi zeribosome ezidayiweyo ukuze zihlolisise izixa ezifakiweyo, kuquka uphando lwezenzululwazi zobisi olusebenzisa iibhendi zeribosome ezidaywe nge-methylene blue ukulungelelanisa izixa. Iimembrane ze-Southern zinokuboniswa ngendlela efanayo, kodwa idayi isekelwe kakhulu njengendlela yokuhlola izixa ezifakiweyo nokudluliselwa kwi-Northern. I-DNA ye-Southern iqhawulwa isakhiwo ngaphambi kokudluliselwa, ngoko amanani obuntununtunu bejeli awasebenzi ngqo kuyo.

Imida yokufumanisa nombala ongemva: apho idayi eluhlaza ingaphumeleli khona

Uthelekiso olungezantsi luqikelelwe ngabom. Ukusebenza kwedayi kuxhomekeke kwipesenti yejeli, kububanzi bebhendi, kwizixhobo zokubona, nakwindlela yokususa idayi. Thatha inani ngalinye njengoqikelelo lobukhulu, kungekhona njengomlinganiselo oqinisekisiweyo.

Ukulawula umbala ongemva kugqiba ukuba uphawu oluthambileyo lunokusetyenziswa na. Idayi engabophelelanga inombala oqinileyo, ngoko umahluko uvela ngokususa idayi engabophelelanga ngaphandle kokukhupha ebopheleleyo. Idayi exineneyo ngakumbi ayifane yongeze ubuntununtunu. Ikakhulu yongeza ixesha lokususa idayi. Qhuba uthotho lwezixa ezixutywe ngokwahlukeneyo usebenzisa kwaloo jeli, imembrane, nekhamera, uze usebenzise kuphela uluhlu apho ubukhulu bomqondiso odityanisiweyo buhlala bulingana nesixa esifakiweyo. I-methylene blue ixhasa ukulungelelaniswa kwezixa ezifakiweyo ngendlela enika uqikelelo lobungakanani. Ayilovavanyo lobungakanani olunoluhlu olubanzi lokulinganisa, kuba umthamo wemembrane, ukusebenza kokudluliselwa, ukuzala, nokususwa kwedayi kuyahluka ukusuka kwenye indlela ukuya kwenye.

Imida yokuhambelana: yintoni esebenzayo emva kokudaya

Ukuklona emva kokudaya ngeblue kubonisiwe. UFlores noogxa bakhe basika iibhendi ze-agarose ezidayiweyo, bafumana i-DNA, bayidibanisa baza bayifaka kwiiseli, bexela ukuklona okungcono kunasemva kwe-ethidium bromide ene-UV. Isizathu esinokwenzeka kukuncipha komonakalo wokukhanya, hayi inzuzo yedayi ngokwayo. Gcina ixesha lokuchanabeka ekukhanyeni okumhlophe lifutshane, uze ucoce i-DNA efunyenweyo ngenkqubo eqhelekileyo yokukhupha i-DNA kwijeli.

Ubungqina be-PCR nokufunda ulandelelwano buncinane ngakumbi. Idayi eseleyo ibophelela kwii-asidi zenukliyiki, ngoko susa idayi uze ucoce ngaphambi kwamanyathelo asebenzisa ii-enzyme. Kulindeleke ukuba i-DNA ecocwe ngokufanelekileyo kwijeli edaywe ngeblue isebenze, kodwa ukunyamezelwa kwedayi edluliselwa ngqo kwiintlobo ezahlukeneyo ze-polymerase nakwiikhemistri zokufunda ulandelelwano akukalinganiswa kakuhle. Musa ukukuthatha njengento eqinisekileyo.

Ihayibridayizeshini yeblot iyahambelana xa ulandelelwano lulandelwa ngokungqongqo: dlulisela, milisela, daya, bhala phantsi iziphumo, susa idayi, wenze ihayibridayizeshini. Olo landelelwano luyilo lukaHerrin noSchmidt. Ukudaya ngaphambi kokudluliselwa, okanye ukutsiba ukususa idayi, yindlela eyahlukileyo kwaye ifuna ukuqinisekiswa kwayo. Emva kokususwa kwedayi, ukususa iprobe ngendlela eqhelekileyo nokuphinda kuvavanywe ngeprobe kuqhubeka njengesiqhelo. Ngokudaya kwinqanaba leseli ngolu sapho lunye lwedayi, nangokuba kutheni indlela yesampulu emiliselweyo ingasetyenziswa ngqo kwiindlela zeeseli eziphilayo, jonga iatlasi yokudaya kwimayikhroskopi.

Ukusika iibhendi kuyasebenza kwi-agarose yokulungiselela ukusetyenziswa kamva nakwii-oligo ze-PAGE ezifunyaniswe ngeblue, ukuba kuphetshwa indlela yokumilisela ngokusisigxina. Ukukhupha ngesixhobo sombane emva kokususa idayi kuyavakala ngokwekhemistri kwisibopheleli esibuyisekayo, kodwa iinkcukacha ezilawulweyo zesixa esifunyenweyo ezijoliswe kwi-methylene blue zinqabile. Susa iityuwa okanye wenze iprecipitation emva kokukhupha.

Iintsilelo eziphindaphindayo

Iibhendi zejeli ezingabonakali kakuhle zidla ngokuthetha ukuba kukho ubunzima obuncinane kakhulu kwidayi ebonisa ngombala, ngakumbi kwiisampulu ekujoliswe kuzo ezimfutshane ezinemisonto enye okanye ze-RNA. Faka isixa esingaphezulu, sebenzisa idayi entsha, wandise ixesha lokudaya ngaphambi kokunyusa ukuxinana, uze uyeke ukususa idayi kwangethuba. Ijeli eluhlaza ngokupheleleyo engenamahluko idla ngokuthetha ukudaya okugqithisileyo okanye ukutshintsha amanzi izihlandlo ezimbalwa kakhulu. Ukuphelelwa ngumbala kweebhendi ze-PAGE yikhemistri elindelekileyo, kungekhona ukusilela kwedayi. Fota ngokukhawuleza. Umbala ongemva ongalinganiyo kwimembrane ubonisa ukusongwa, amaqamza, imizila yeminwe, ukushukunyiswa okungalinganiyo, okanye ukudluliselwa okungalinganiyo. Musa ukukutolika oko njengezixa ezingalinganiyo ezifakiweyo ngaphandle kovavanyo lolawulo lwehayibridayizeshini. Ipateni yeribosome ebuthathaka nenwenwileyo inokuthetha ukonakala, kodwa isenokuthetha ukudluliselwa okungaphelelanga, isixa sonke se-RNA esincinane kakhulu (iindlela eziqhelekileyo zithwala ukusuka kwii-micrograms ezimbalwa ukuya kumashumi azo), okanye ukususa idayi ngokugqithisileyo. Idayi ibonisa i-RNA yeribosome eninzi. Ayibonisi umbhalo othile we-RNA. Loo msebenzi ngoweprobe.

Ukhuseleko nokuphatha ngaphandle kwamabango agqithisileyo

I-methylene blue kulula ngakumbi ukuyiphatha kune-ethidium bromide, kodwa ayiyonto engenabungozi. Iphepha langoku leenkcukacha zokhuseleko le-MilliporeSigma lihlela idayi njengeyetyhefu ebukhali xa iginyiwe, iCategory 4, eyingozi xa iginyiwe, efanele ukulahlwa ngendlela evunyiweyo yenkunkuma ngokwe-SDS ye-methylene blue ye-Sigma-Aldrich. I-ethidium bromide inezilumkiso ezinamandla ngakumbi zokonakalisa imathiriyeli yemfuza, kuquka iziphumo ezikrokrelekayo kwiiseli zokuzala kwi-SDS ye-ethidium bromide ye-Sigma-Aldrich, yaye iijeli nezinyibilikiso zayo ziqhele ukwahlulwa njengenkunkuma yemichiza eyingozi, njengoko kuphawulwe kwisikhokelo seedayi zee-asidi zenukliyiki se-Thermo Fisher. Landela i-SDS nemithetho yeziko kuzo zombini. Gcina idayi eyomileyo ivalwe ngokuqinileyo kwaye yomile, phinda usebenzise idayi yokusebenza kuphela ngendlela evunyelwa yiprothokholi, gcina iimembrane ezidayiweyo zomile phakathi kwamaphepha esihluzi, uze ufote iijeli ngaphambi kokuba iibhendi zisasazeke ziphele.

Khumbula lo musho wasetafileni yelabhoratri. Iijeli zivavanya ukubonakala kwezixa eziphezulu phantsi kokukhanya okumhlophe. Iimembrane zivavanya ukudluliselwa nezixa ezifakiweyo ngaphambi kokuba iprobe yenze umsebenzi onobuntununtunu obuphezulu. Idayi enye isebenzela zombini, ngobuntununtunu obuphakathi, ukubophelela okubuyisekayo, nolandelelwano olungqongqo lwamanyathelo.