I-methylene blue yokudaya i-DNA ne-RNA: ukusetshenziswa kumajeli nasolwelwesini

Umfundi wenza i-electrophoresis ngejeli ye-agarose, ayidaye nge-methylene blue, angaboni lutho lapho bekulindeleke khona umugqa ogqamile. Endaweni yokusebenza eseduze, umcwaningi oseneziqu zobudokotela udaya ulwelwesi lwe-Northern ngokusebenzisa ibhodlela elifanayo likadayi oluhlaza okwesibhakabhaka, athole imigqa ecacile ye-ribosomal ngemizuzu emithathu. Umfundi uphetha ngokuthi ijeli ihlulekile. Ijeli ayihlulekanga. Le misebenzi emibili ibihlukene, futhi udayi ofanayo uyiphendula ngokuzwela okuhlukene, ngengemuva elihlukene, nangemithetho ehlukene yezinyathelo ezilandelayo.

Udayi we-DNA we-methylene blue nokudaya i-RNA nge-methylene blue kusebenzisa ikhemistri efanayo kodwa kunezinhloso ezingafani. Ejelini, udayi ubonisa ukuthi kungakanani i-nucleic acid engaphakathi kwesakhiwo esinezimbotshana. Olwelwesini, ubonisa ukuthi ukudluliswa kusebenzile yini nokuthi amasampula afakwe ngokulinganayo yini emizileni. Uma lo mehluko usucacile, izinqubo, imikhawulo yokuthola, nemikhawulo yokuhambisana kuyaqondakala.

Impendulo emfushane

I-methylene blue ingudayi oyisicaba one-charge ephozithivu, obopha ama-nucleic acid ngaphandle kwezibopho ze-covalent futhi ngendlela ehlehlisekayo, ngenhlanganisela yokukhangana kwama-charge ne-phosphate, ukungena phakathi kwamapheya ama-base, nokubopha emseleni omncane. Yilokho kuhlehliseka okubalulekile. Imigqa ingadaywa, ibukwe ngokukhanya okubonakalayo, iqoshwe, bese kususwa udayi noma ihlanzwe ukuze kwenziwe i-cloning, i-hybridization, noma iphinde ihlolwe nge-probe.

Ekudayeni i-DNA enemicu emibili ejelini ye-agarose nge-methylene blue, ingxube evamile esheshayo ingu-0.02 percent w/v (200 micrograms per mL) emanzini, kudaywe cishe imizuzu engu-15 bese kususwa udayi ngamanzi cishe imizuzu engu-15, kubukwe ebhokisini lokukhanya okumhlophe. Olwelwesini, ingxube yakudala ye-Northern blot ingu-0.02 kuya ku-0.04 percent kadayi ku-sodium acetate eduze kwe-pH 5.2, kudaywe imizuzu embalwa ngemva kokudluliswa nokugxiliswa kwesampula, bese kuhlanjululwa ngamanzi futhi kususwe udayi ngokuphelele ngaphambi kwe-hybridization. Ukuzwela kulinganiselwe: amashumi kuya emakhulwini ama-nanogram ngomugqa ngamunye wejeli ngaphansi kwezimo ezinhle, kanti iziqondiso eziqaphile zisho u-0.5 kuya ku-1 microgram. Lokho kuphakeme ngezilinganiso eziningana zokuphindaphinda ngeshumi kunemikhawulo yodayi be-ethidium bromide no-SYBR. Olwelwesini akukho mkhawulo owodwa owusizo wokuthola i-transcript eyodwa ngoba udayi ubonisa ikakhulukazi i-ribosomal RNA eningi kanye nenani eliphelele lesampula elifakiwe. Ungasebenzisi isixazululo sesengezo esithengiswa ezitolo esikhundleni sikadayi kunoma iyiphi yalezi zingxube. Izindlela eziqinisekisiwe zithatha ngokuthi kusetshenziswa udayi wezinga lokuhlaziya, ama-buffer achazwe ngokucacile, nokuphathwa okungenayo i-RNase lapho kuhileleke khona i-RNA.

Ukubopha kusebenza kanjani, nokuthi kungadukisa kuphi

Imodeli ewusizo inezingxenye ezintathu. Okokuqala, uhlelo lwamasongo olune-charge ephozithivu lukhangwa umgogodla we-phosphate one-charge enegethivu. Ucwaningo lwe-spectroscopy luthola ukuthi lokhu kukhangana kwama-charge kubusa lapho usawoti umncane, kuyilapho ukumodela nokuhlola ukunyakaza kwama-molecule kugcina ukungena phakathi kwamapheya ama-base nokubopha emseleni omncane njengezindlela ezizinzile ze-DNA enemicu emibili, njengoba kufingqiwe ocwaningweni lwe-spectroscopy lokubopha kwe-methylene blue ku-DNA. Okwesibili, kukhona ukuthambekela okuthile ku-GC, njengoba ukumodela kuvuna izinyathelo ze-CpG ne-GpC, kanti ukuhlola kubika ukubopha okuthanda ama-duplex anothe nge-GC ocwaningweni lokuthambekela kokungena kwe-methylene blue phakathi kwamapheya ama-base, kodwa ama-duplex e-AT nawo ayabopha. Ukudaya okuvamile akuqondile ekulandelaneni okuthile. Okwesithathu, i-DNA enomucu owodwa ne-RNA kubopha ikakhulukazi ngokukhangana kwama-charge nangokupakisheka kwama-base afinyelelekayo nezifunda ezinemicu emibili, yingakho amandla okudaya ehluka ngobude, ukulandelana, nesakhiwo sesibili.

Kulandela imiphumela emibili. Ngenxa yokuthi ukubopha akusebenzisi izibopho ze-covalent, udayi uyagezeka uphume. Imigqa ye-polyacrylamide iyafiphala ngokushesha ngaphandle kokuba igxiliswe ngenhloso, futhi kukhona indlela ekhethekile yokugxilisa unomphela ngenxa yokuthi ukudaya okuvamile kungokwesikhashana, njengoba kuchazwe ngu-Soto no-Draper kumajeli e-PAGE. Ngenxa yokuthi udayi uyisivuseleli sezinqubo zamakhemikhali ngokukhanya, ukukhanyisa i-DNA edayiwe isikhathi eside ngokukhanya okukhulu lapho kukhona umoya-mpilo kungabangela i-oxidation ye-guanine futhi ekugcineni kunqamule imicu, indlela echazwe ngokuningiliziwe ocwaningweni lwe-oxidation ye-guanine evuselelwa yi-methylene blue. Ukubuka isikhashana ngokukhanya okumhlophe kugwema ukulimala kwe-UV okwaziwayo ezinqubweni zakudala ze-ethidium bromide, futhi leyo nzuzo ibalulekile ekubuyiseni i-DNA ukuze isetshenziswe. Ukukhanya okubonakalayo kuthambile, kodwa akusho ukuthi akunangozi. Gcina isikhathi sokukhanyisa sifushane uma i-DNA izosetshenziselwa i-cloning. Ukuze uthole ikhemistri yokukhanya ebanzi esekela lesi sexwayiso, bheka izindlela ezimbili kukhemistri yokukhanya ye-methylene blue. Ukuze ufunde ngababili be-redox nezimo zama-charge ezichaza ukuthi kungani uhlobo olwenziwe i-oxidation lubopha kodwa olwenziwe i-reduction lungabophi, bheka ikhemistri yesakhiwo nezakhiwo ze-methylene blue.

Umsebenzi wokuqala: ukudaya ngaphakathi kwamajeli

Ukudaya ijeli kuphendula umbuzo owodwa: ingabe kunama-nucleic acid anele kulo mugqa ukuze ubonakale ngeso. I-DNA enemicu emibili ku-agarose iyisimo esicwaningwe kahle kakhulu. Indlela yokuqala yokudaya ijeli nge-methylene blue ka-Flores nozakwabo yaqinisekisa inqubo esheshayo engu-0.02 percent ku-agarose engu-1 percent futhi yabonisa ukubuyiswa okuphumelelayo ne-cloning ngemva kwalokho. Inqubo efanayo yomphakeli itholakala ekhasini lomkhiqizo we-methylene blue le-IBI Scientific, elibeka ingxube yolwelwesi engu-0.04 percent nendlela esheshayo yejeli eceleni komunye nomunye.

Inqubo esebenzisekayo:

1. Yenza i-electrophoresis yejeli ngendlela evamile. Daya ngemva kwe-electrophoresis, hhayi phakathi nayo, ngaphandle uma ukudluliswa noma ukuhlukaniswa sekuqinisekisiwe ngendlela eguquliwe.
2. Cwilisa ku-methylene blue engu-0.02 percent w/v emanzini acwengwe ngokubiliswa nokufingqa, unyakazise ngobumnene, cishe imizuzu engu-15.
3. Susa udayi ngokushintsha amanzi izikhathi eziningana, cishe imizuzu engu-15 isiyonke, kuze kwehle umbala wengemuva lesakhiwo sejeli futhi imigqa igqame.
4. Thatha isithombe ngokushesha kusikhanyisi esinokukhanya okumhlophe.

I-agarose ne-polyacrylamide ziziphatha ngendlela ehlukene. I-agarose igcina udayi ngaphakathi kwesakhiwo sayo, ngakho ukususa udayi kuyisinyathelo esihamba kancane. Amajeli azacile e-polyacrylamide adaya ngokushesha kodwa aphinde alahlekelwe udayi ngokushesha, okusiza ukusika imigqa ukuze isetshenziswe kodwa kuphazamise amarekhodi agcinelwa isikhathi eside. Ama-oligonucleotide enziwe ngokwenziwa ku-polyacrylamide angatholakala ngengxube efanayo engu-0.02 percent ngemizuzu engu-20 kuya ku-30 ngokugeza ngamanzi ngokuphindaphindiwe, kulandelwa izindlela ze-PAGE ezimisiwe zohlobo lwe-Current Protocols. Kubikwe izindlela ezixutshwe kakhulu, eziseduze no-0.0002 percent ku-TBE exutshiwe, zokulungisa ama-oligo emahoreni amaningana, kodwa ubufakazi obuzisekelayo buncane.

Umehluko phakathi kwe-DNA ne-RNA ubalulekile. I-DNA enemicu emibili ibopha ngamandla kakhulu ngendlela ehlelekile. I-RNA ne-DNA enomucu owodwa kuyadaya, kodwa ngokubuthaka nangendlela engaqageleki kahle. Kumajeli e-RNA e-formaldehyde agarose, amalabhorethri amaningi ayakweqa ukudaya ngodayi oluhlaza okwesibhakabhaka ngaphakathi kwejeli, aqale adlulisele i-RNA olwelwesini, bese edaya lapho. Lokho kushintsha isignali ebuthaka yejeli kube isignali ecace kakhudlwana yolwelwesi kanye nerekhodi lokufakwa kwamasampula.

Umsebenzi wesibili: ukudaya olwelwesini

Ukudaya ulwelwesi kuphendula umbuzo ohlukile: ingabe ukudluliswa kusebenzile, futhi ingabe imizila ingaqhathaniswa. Ukusetshenziswa kwakudala kungudayi we-Northern blot ohlehlisekayo ka-Herrin no-Schmidt, owawuqale ungu-0.04 percent kadayi ku-50 mM sodium acetate ku-pH 5.0 imizuzu engu-2 kuya ku-3, bese kuhlanjululwa ngamanzi, futhi ungasuswa nge-SDS exutshiwe ku-SSPE ngaphambi kwe-hybridization.

Indlela yesimanje esetshenziswa kabanzi ilandela lezi zinyathelo:

1. Dlulisela i-RNA noma i-DNA ehlukaniswe imicu ku-nylon noma ku-nitrocellulose, bese uyigxilisa ngokuxhumanisa nge-UV noma ngokuyibhaka ukuze isampula ingalahleki ngesikhathi sokudaya.
2. Akuphoqelekile kodwa kuvamile: manzisa ulwelwesi ku-acetic acid engu-5 percent cishe imizuzu engu-15.
3. Daya imizuzu engu-5 kuya ku-10 ku-methylene blue engu-0.02 kuya ku-0.04 percent ku-0.3 kuya ku-0.5 M sodium acetate eduze kwe-pH 5.2.
4. Hlanza bese ususa udayi ngamanzi imizuzu engu-5 kuya ku-15, qopha isithombe, bese ususa udayi osele ngaphambi kwe-hybridization.

I-nylon ene-charge ephozithivu iyona esetshenziswa kakhulu emsebenzini we-Northern. I-nitrocellulose nayo isebenza ngenqubo efanayo ye-acetic acid ne-acetate. Isithombe sisetshenziselwa ukuqinisekisa ukudluliswa, ukuqhathanisa imigqa ye-28S ne-18S (noma ye-23S ne-16S ku-RNA yamagciwane), ukumaka izitebhisi nezilinganiso, nokugcina irekhodi elilinganiselwe lokwenza amanani e-RNA ephelele aqhathaniseke. Izindlela ezishicilelwe ze-Northern zikala imigqa ye-ribosomal edayiwe ukuze zihlole ukufakwa kwamasampula, kuhlanganise nomsebenzi wesayensi yobisi owasebenzisa imigqa ye-ribosomal edaywe nge-methylene blue ukwenza amanani aqhathaniseke. Ulwelwesi lwe-Southern lungabonwa ngendlela efanayo, kodwa udayi waziwa kakhulu njengendlela yokuhlola ukufakwa kwamasampula nokudluliswa kwe-Northern, kanti i-DNA ye-Southern ihlukaniswa imicu ngaphambi kokudluliswa, ngakho izibalo zokuzwela zejeli azisebenzi ngokuqondile lapho.

Imikhawulo yokuthola nengemuva: lapho udayi oluhlaza okwesibhakabhaka usilela khona

Ukuqhathanisa okungezansi kulinganiselwe ngenhloso. Ukusebenza kukadayi kuncike kwiphesenti lejeli, ububanzi bomugqa, izinsiza zokubuka, nokulandela kahle indlela yokususa udayi. Thatha inombolo ngayinye njengesilinganiso sobukhulu, hhayi njengemininingwane eqinisekisiwe yokusebenza.

Ukulawula ingemuva kunquma ukuthi isignali elinganiselwe ingasetshenziswa yini. Udayi ongaboshiwe unombala ogqamile, ngakho umehluko uvela ekususeni udayi ongaboshiwe ngaphandle kokususa oboshiwe. Udayi ominyene kakhulu awuvamile ukwengeza ukuzwela. Ikakhulukazi wengeza isikhathi sokususa udayi. Hlola uchungechunge lokuxutshwa kwejeli, ulwelwesi, nekhamera ozisebenzisa ngempela, bese usebenzisa kuphela ibanga lapho ukuminyana kombala okuhlanganisiwe kuhlala kuhambisana ngokulingana nenani elifakiwe. I-methylene blue isekela ukwenziwa kwamanani okufakwa kwamasampula aqhathaniseke ngesilinganiso esingaphelele. Ayisona isivivinyo sokulinganisa esinebanga elikhulu lokulinganisa ngoba umthamo wolwelwesi, ukusebenza kahle kokudluliswa, ukugcwala, nokususwa kukadayi kuyahluka emzileni ngamunye.

Imikhawulo yokuhambisana: yini esebenza ngemva kokudaya

I-cloning ngemva kokudaya ngoluhlaza okwesibhakabhaka ibonisiwe. U-Flores nozakwabo basika imigqa ye-agarose edayiwe, babuyisa i-DNA, bayihlanganisa nge-ligation futhi bayifaka kumaseli nge-transformation, babika i-cloning engcono kunangemva kwe-ethidium bromide ene-UV. Isizathu esingaba khona ukulimala okuncane kokukhanya, hhayi inzuzo evela kudayi uqobo. Gcina ukuchayeka ekukhanyeni okumhlophe kufushane bese uhlanza i-DNA ebuyisiwe ngendlela evamile yokukhipha i-DNA ejelini.

Ubufakazi be-PCR nokunquma ukulandelana buncane kakhulu. Udayi osele ubopha ama-nucleic acid, ngakho susa udayi bese uhlanza ngaphambi kwezinyathelo zama-enzyme. I-DNA ehlanzwe kahle evela ejelini edaywe ngoluhlaza okwesibhakabhaka kulindeleke ukuthi isebenze, kodwa ukubekezelelwa kukadayi osele kuma-polymerase nasezinhlotsheni zekhemistri zokunquma ukulandelana akukalinganiswa kahle. Ungakuthathi njengokuqinisekisiwe.

I-hybridization ye-blot iyahambisana uma uhlelo lwezinyathelo lulandelwa ngokuqinile: dlulisa, gxilisa, daya, qopha, susa udayi, yenza i-hybridization. Lolo chungechunge luyindlela ka-Herrin no-Schmidt. Ukudaya ngaphambi kokudlulisa, noma ukweqa ukususa udayi, kuyindlela ehlukile futhi kudinga ukuqinisekiswa kwakho. Ngemva kokususa udayi, ukususa ama-probe ngendlela evamile nokuphinda kuhlolwe ngama-probe kuqhubeka ngokujwayelekile. Ukuze ufunde ngokudaya ezingeni lamaseli ngomndeni ofanayo kadayi nokuthi kungani indlela yezendlalelo ezigxilisiwe ingasebenzi ngokuqondile ezindleleni zamaseli aphilayo, bheka i-atlas yokudaya ye-microscopy.

Ukusika imigqa kuyasebenza ku-agarose elungiselelwa ukubuyisa isampula nakuma-oligo e-PAGE atholwe ngodayi oluhlaza okwesibhakabhaka, uma kugwenywa indlela yokugxilisa unomphela. Ukukhipha ngogesi ngemva kokususa udayi kuyenzeka ngokwekhemistri uma udayi ebopha ngendlela ehlehlisekayo, kodwa idatha elawulwayo yobuningi obubuyisiwe eqondene ne-methylene blue incane. Susa usawoti noma wenze isampula ibe yinsalela eqinile ngemva kokuyikhipha.

Izinkinga eziphindaphindayo

Imigqa yejeli efiphele ngokuvamile isho ukuthi isisindo sincane kakhulu ukuze sibonwe ngodayi wombala, ikakhulukazi kuma-target amafushane anomucu owodwa noma e-RNA. Faka okuningi, sebenzisa udayi omusha, yelula isikhathi sokudaya ngaphambi kokwandisa ukugxila, futhi yeka ukususa udayi kusenesikhathi. Ijeli eluhlaza okwesibhakabhaka ngokuphelele engenawo umehluko ogqamile ngokuvamile isho ukudaya ngokweqile noma ukushintsha amanzi izikhathi ezimbalwa kakhulu. Ukufiphala kwemigqa ye-PAGE kuyikhemistri elindelekile, hhayi ukuhluleka kokudaya. Thatha isithombe ngokushesha. Ingemuva lolwelwesi elingalingani likhomba ukugoqeka, amabhamuza, izimpawu zeminwe, ukunyakaziswa okungalingani, noma ukudluliswa okungalingani ezindaweni ezahlukene. Ungakuhumushi njengokufakwa kwamasampula okungalingani ngaphandle kokuhlola kokulawula kwe-hybridization. Iphethini ye-ribosomal ebuthaka nenwebekile ingasho ukuwohloka, kodwa futhi ingasho ukudluliswa okungaphelele, i-RNA ephelele encane kakhulu (imizila evamile iqukethe ama-microgram ambalwa kuya emashumini), noma ukususa udayi ngokweqile. Udayi ubonisa i-ribosomal RNA eningi. Awubonisi i-transcript ethile. Lowo msebenzi ungowe-probe.

Ukuphepha nokuphatha ngaphandle kokweqisa izimangalo

I-methylene blue ilula ukuyiphatha kune-ethidium bromide, kodwa ayiyona engenangozi. Ishidi lamanje lemininingwane yokuphepha le-MilliporeSigma lihlukanisa udayi njengonobuthi obusheshayo uma ungena ngomlomo, Isigaba 4, uyingozi uma ugwinyiwe, futhi kufanele ulahlwe ngendlela egunyaziwe yokulahla imfucuza ngokwe-SDS ye-methylene blue ye-Sigma-Aldrich. I-ethidium bromide inezexwayiso ezinamandla kakhulu zokulimaza izinto zofuzo, kuhlanganise nemiphumela esolwayo kumaseli okuzala ku-SDS ye-ethidium bromide ye-Sigma-Aldrich, futhi amajeli nezixazululo zayo kuvame ukuhlukaniswa njengemfucuza yamakhemikhali eyingozi njengoba kuphawulwe esiqondisweni sodayi bama-nucleic acid se-Thermo Fisher. Landela i-SDS nemithetho yesikhungo kokubili. Gcina udayi owomile uvalwe ngokuqinile futhi womile, phinda usebenzise udayi osebenzayo kuphela njengoba inqubo ivumela, gcina ulwelwesi oludayiwe lomile phakathi kwamashidi okuhlunga, futhi uthathe izithombe zamajeli ngaphambi kokuba imigqa isakazeke.

Khumbula lo musho wasetafuleni lokusebenza. Amajeli ahlola ukubonakala lapho kufakwe inani elikhulu ngaphansi kokukhanya okumhlophe. Ulwelwesi luhlola ukudluliswa nokufakwa kwamasampula ngaphambi kokuba i-probe yenze umsebenzi odinga ukuzwela okukhulu. Udayi owodwa usebenza kukho kokubili, ngokuzwela okulinganiselwe, ukubopha okuhlehlisekayo, nokulandelana kwezinyathelo okuqinile.