Tuhinga
Te kikorangi metilēna hei whakawaikano i te DNA me te RNA: ngā whakamahinga ki ngā tīre me ngā kiriuhi
Ka whakahaere tētahi ākonga i tētahi tīre agarose, ka whakawaikano ki te kikorangi metilēna, ā, kāore e kitea he mea i te wāhi i tūmanakohia ai he pae kanapa. I te wāhi mahi i tōna taha, ka whakawaikano tētahi kairangahau whai muri i te tohu kairangi i tētahi kiriuhi Northern ki taua pounamu kikorangi anō, ā, ka puta he pae ribosome koi i roto i te toru meneti. Ka whakatau te ākonga kua rahua te tīre. Kāore te tīre i rahua. He rerekē ngā mahi e rua, ā, he rerekē te tairongo, te papamuri, me ngā ture mō ngā mahi ka whai mai i te whakamahinga o taua waikano kotahi.
He ōrite te matū o te waikano DNA kikorangi metilēna me te whakawaikano RNA kikorangi metilēna, engari he rerekē te kaupapa. I roto i te tīre, ka whakaatu te waikano i te nui o te waikawa karihi kei roto i tētahi hanganga kōputaputa. I runga i te kiriuhi, ka whakaatu mēnā i angitu te whakawhiti, ā, mēnā i ōrite te utanga o ngā ara. Kia mārama taua wehenga, ka mārama hoki ngā kawa, ngā paenga kite, me ngā paenga hototahi.
Te whakautu poto
He waikano katote tōrunga, papatahi hoki te kikorangi metilēna, e here ana ki ngā waikawa karihi mā ngā here kore-kōwaro e taea ana te wetewete. He ranunga tēnei o te kukume hiko-pateko ki te phosphate, te kuhu i waenga i ngā takirua pūtake, me te here ki te awa iti. Ko taua āheinga ki te wetewete te tino take. Ka taea te whakawaikano i ngā pae, te tiro mā te aho kitea, te tuhi whakaahua, kātahi ka tango i te waikano, ka whakapurenga rānei mō te whakapūruatanga, te whakahononga, te whakamātautau anō ki te pūoko rānei.
Mō te whakawaikano tīre agarose ki te kikorangi metilēna mō te DNA aho-rua, ko te tohutaka tere paerewa he 0.02 percent w/v (200 micrograms per mL) ki te wai, he 15 meneti pea te whakawaikano me te 15 meneti pea te tango waikano ki te wai, ka tirohia ki runga i tētahi pouaka rama mā. Mō ngā kiriuhi, ko te tohutaka Northern blot tuku iho he waikano 0.02 ki te 0.04 percent ki te sodium acetate tata ki te pH 5.2, he meneti torutoru te whakawaikano i muri i te whakawhiti me te whakamau, kātahi ka horoi ki te wai, ka tango katoatia te waikano i mua i te whakahononga. He āhua iti te tairongo: he tekau ki te rau nanogram mō ia pae tīre i raro i ngā tikanga pai, ā, e ai ki ngā aratohu tūpato, he 0.5 ki te 1 microgram. He maha ngā taumata whakareatanga tekau teitei ake tērā i ngā waikano ethidium bromide me SYBR. Mō ngā kiriuhi, kāore he paenga kite kotahi e whai tikanga ana mō tētahi tāruatanga RNA, nā te mea ko te RNA ribosome nui me te katoa o ngā matū kua utaina ngā mea matua ka whakaaturia e te waikano. Kaua e whakakapi i tētahi o ēnei tohutaka ki te wairewa tāpiringa e hokona ana ki te marea. E whakapae ana ngā tikanga kua whakamanahia ka whakamahia he waikano taumata tātari, he wairewa ārai pH kua tautuhia, me te whakahaere kore-RNase ina whai wāhi te RNA.
Te āhua o te here, me ngā wā ka whakapohehe
E toru ngā wāhanga o te tauira whai hua. Tuatahi, ka kukumea te pūnaha mowhiti whai utanga tōrunga ki te tuarā phosphate whai utanga tōraro. E ai ki ngā rangahau tūāwhiorangi, ko tēnei kukume hiko-pateko te mea matua ina iti te tote. Heoi, e whakaatu tonu ana ngā whakatauira me ngā rangahau nekeneke rāpoi i te kuhu ki waenga me te here ki te awa iti hei āhua pūmau mō te DNA aho-rua, e whakarāpopototia ana i tētahi rangahau tūāwhiorangi mō te here o te kikorangi metilēna ki te DNA. Tuarua, he hiahia paku ki te GC: e pai ake ana ngā takahanga CpG me GpC i ngā whakatauira, ā, e pūrongo ana ngā whakamātautau i te here pai ake ki ngā aho-rua whai GC nui i ngā mahi mō ngā manakohanga kuhu o te kikorangi metilēna. Engari ka here hoki ngā aho-rua AT. Kāore te whakawaikano auau e whāiti ana ki tētahi raupapa. Tuatoru, ko te nuinga o te here ki te DNA aho-kotahi me te RNA mā te kukume hiko-pateko me te tāpae ki ngā pūtake e wātea ana me ngā wāhanga aho-rua. Koia te take e rerekē ai te kaha whakawaikano i runga i te roa, te raupapa, me te hanganga tuarua.
E rua ngā hua ka whai mai. Nā te mea kāore he here kōwaro, ka horoia atu te waikano. Ka tere memeha ngā pae polyacrylamide ki te kore e āta whakamaua. He tikanga whakamau pūmau motuhake anō, nā te mea he rangitahi noa te whakawaikano noa, e whakaahuatia ana e Soto rāua ko Draper mō ngā tīre PAGE. Nā te mea he whakahohe-aho te waikano, ka taea e te whakamārama kanapa mō te wā roa i te DNA kua whakawaikanotia, me te hāora i reira, te whakahāora i te guanine, ā, i te mutunga, te motu i ngā aho. Ka whakamāramatia tēnei tikanga i ngā mahi mō te whakahāoratanga guanine i whakahohea e te kikorangi metilēna. Mā te tiro poto ki te rama mā ka karohia te tūkinotanga UV e mōhiotia whānuitia ana i ngā tukanga ethidium bromide tuku iho, ā, he tino painga tērā mō te whakahokinga hei whakamahi anō. He ngāwari ake te aho kitea, engari ehara i te mea kāore he kino. Kia poto te whakamārama ina whakamahia te DNA mō te whakapūruatanga. Mō ngā tauhohenga matū-aho whānui kei muri i taua tūpato, tirohia ngā ara e rua i te matū-aho o te kikorangi metilēna. Mō te takirua whakahāora-whakaheke me ngā āhua utanga e whakamārama ana i te here o te āhua whakahāora, engari kāore te āhua whakaheke e here, tirohia te matū o te hanganga me ngā āhuatanga o te kikorangi metilēna.
Te mahi tuatahi: te whakawaikano i roto i ngā tīre
Kotahi te pātai e whakautua ana e te whakawaikano tīre: he nui rānei te waikawa karihi i tēnei pae kia kitea e te kanohi? Ko te DNA aho-rua i roto i te agarose te take kua tino rangahautia. Nā te tikanga tīre kikorangi metilēna taketake a Flores me ōna hoa i whakamana te kawa tere 0.02 percent ki runga i te agarose 1 percent, me te whakaatu i te angitu o te whakahokinga me te whakapūruatanga whai muri. Kei te whārangi hua kikorangi metilēna a IBI Scientific taua kawa kaiwhakarato anō, e whakarārangi ngātahi ana i te tohutaka kiriuhi 0.04 percent me te tikanga tīre tere.
Te tukanga ā-mahi:
- Whakahaerehia te tīre i te tikanga noa. Whakawaikanotia i muri i te hiko-whakawehe, kaua i te wā e haere ana, ki te kore te whakawhiti, te kaha wehe rānei, e whakamanahia mō tētahi tikanga kua whakarerekētia.
- Rumakina ki te kikorangi metilēna 0.02 percent w/v i roto i te wai māturu, me te rūrū ngāwari, mō te 15 meneti pea.
- Tangohia te waikano mā te huri i te wai i ētahi wā, mō te 15 meneti pea te katoa, kia heke te papamuri o te hanganga, kia mārama ngā pae.
- Hopukina wawetia he whakaahua ki runga i tētahi pūrere whakamārama rama mā.
He rerekē te whanonga o te agarose me te polyacrylamide. Ka pupuri te agarose i te waikano i roto i tōna hanganga, nō reira ko te tango waikano te wāhanga pōturi. Ka tere whakawaikanotia ngā tīre polyacrylamide angiangi, engari ka tere hoki te ngaro o te waikano. He āwhina tēnei ki te tapahi hei whakamahi anō, engari he raruraru mō ngā pūkete pūranga. Ka taea te kimi i ngā oligonucleotide waihanga i roto i te polyacrylamide mā taua tohutaka 0.02 percent anō i roto i te 20 ki te 30 meneti, me ngā horoinga wai tukurua, e whai ana i ngā tikanga PAGE momo Current Protocols kua whakapūmautia. Kua pūrongotia ētahi tikanga tino waimeha, tata ki te 0.0002 percent i roto i te TBE waimeha, mō ngā oligo hei whakamahi anō i roto i ētahi hāora, engari he ngoikore ake te tūāpapa taunakitanga.
He mea nui te rerekētanga o te DNA me te RNA. Ko te DNA aho-rua te mea e whakaputa ana i te here ā-hanganga kaha rawa. Ka whakawaikanotia te RNA me te DNA aho-kotahi, engari he ngoikore ake, he uaua ake hoki te matapae. Mō ngā tīre RNA agarose formaldehyde, kāore te nuinga o ngā taiwhanga e whakawaikano kikorangi ki roto i te tīre; ka whakawhitia kē te RNA ki tētahi kiriuhi i te tuatahi, kātahi ka whakawaikanotia ki reira. Mā tēnei nekehanga ka whakakapia te tohu tīre ngoikore ki tētahi tohu kiriuhi koi ake, me tētahi pūkete utanga.
Te mahi tuarua: te whakawaikano ki runga i ngā kiriuhi
He pātai kē tā te whakawaikano kiriuhi e whakautu: i angitu rānei te whakawhiti, ā, ka taea rānei te whakataurite i ngā ara? Ko te whakamahinga tuku iho ko te waikano Northern blot whakahoki a Herrin rāua ko Schmidt. I te tīmatanga he waikano 0.04 percent i roto i te sodium acetate 50 mM i te pH 5.0, mō te 2 ki te 3 meneti, me te horoi ki te wai. Ka taea te tango mā te SDS waimeha i roto i te SSPE i mua i te whakahononga.
Ko tēnei te tikanga hou e whakamahia whānuitia ana:
- Whakawhitia te RNA, te DNA kua wetekina tōna hanganga rānei, ki te nylon, ki te nitrocellulose rānei, kātahi ka whakamau mā te here-whakawhiti UV, mā te tunu rānei, kia kore ai te tauira e ngaro i te whakawaikanotanga.
- He kōwhiringa, engari he mahi noa: whakamakūkūtia te kiriuhi ki te waikawa acetic 5 percent mō te 15 meneti pea.
- Whakawaikanotia mō te 5 ki te 10 meneti ki te kikorangi metilēna 0.02 ki te 0.04 percent i roto i te sodium acetate 0.3 ki te 0.5 M, tata ki te pH 5.2.
- Horoia, ka tango i te waikano ki te wai mō te 5 ki te 15 meneti, tuhia he pūkete, kātahi ka tango i te toenga waikano i mua i te whakahononga.
Ko te nylon whai utanga tōrunga te mea matua i ngā mahi Northern. Ka whai hua hoki te nitrocellulose mā taua tukanga waikawa acetic me te acetate anō. Ka whakamahia te whakaahua hei whakaū i te whakawhiti, hei whakataurite i ngā pae 28S me 18S (23S me 16S rānei mō te RNA huakita), hei tohu i ngā arawhata me ngā paerewa, ā, hei pupuri i tētahi pūkete whakaritenga āhua noa mō te RNA katoa. Ka ine ngā tikanga Northern kua whakaputaina i ngā pae ribosome kua whakawaikanotia hei arowhai utanga. Kei roto ko ngā rangahau pūtaiao miraka i whakamahi i ngā pae ribosome kikorangi metilēna hei whakaritenga. Ka taea te whakakite i ngā kiriuhi Southern mā taua tikanga anō, engari kua kaha kē atu te whakamahinga o te waikano hei arowhai utanga me te whakawhiti Northern. Waihoki, ka wetekina te hanganga o te DNA Southern i mua i te whakawhiti, nō reira kāore ngā tau tairongo tīre e hāngai tika atu.
Ngā paenga kite me te papamuri: ngā wāhi e ngoikore ai te kikorangi
He whakataurite āwhiwhi noa te mea kei raro nei, i āta whakaritea pēneitia. Ka whakawhirinaki te mahi a te waikano ki te ōrau o te tīre, te whānui o ngā pae, ngā taputapu whatu, me te tika o te tango waikano. Whakaarohia ia tau hei tohu taumata whakareatanga tekau, kaua hei whakaritenga pū.
| Te waikano me te whakatakotoranga | Te paenga kite āwhiwhi mō ia pae | Te whakamāramatanga |
|---|---|---|
| Kikorangi metilēna, dsDNA i roto i te agarose | 40 ki te 250 ng ina arotauhia; 0.5 ki te 1 microgram hei whakatau tūpato | He pai mō ngā utanga hei whakamahi anō me ngā tīre whakaako. He ngoikore mō ngā pae tino iti. |
| Kikorangi metilēna, ssDNA poto, RNA, oligo rānei i roto i te PAGE | Tata ki te 0.1 ki te 0.5 microgram | Ka whakawhirinaki ki te roa me te hanganga. Kāore he paenga whānui kotahi. |
| Ethidium bromide, dsDNA agarose | Tata ki te 1 ki te 10 ng | Tata ki te whā whakareatanga te tairongo ake i te kikorangi kua arotauhia i te whakataurite a Flores. Me whakamahi UV. |
| SYBR Green I, dsDNA | Tata ki te 60 pg | Me whakamahi whakaahua kōrekoreko. |
| SYBR Gold, DNA me te RNA | Tata ki te 25 pg | Neke atu i te tekau whakareatanga te tairongo ake i te ethidium bromide i ētahi whakatakotoranga. |
| Waikano hiriwa, ngā waikawa karihi PAGE | Tata ki te 0.3 ki te 0.9 ng; kua pūrongotia te 100 pg RNA i tētahi raupapa kikorangi-ki-te-hiriwa kua arotauhia | He tino tairongo, engari he matatini te tukanga matū, ā, he ngoikore mō te whakahokinga. |
| Kikorangi toluidine, RNA me ngā oligo PAGE | He rau ng ngā rahinga mahi, tata ki te 250 ng kua tuhia | Ka whakamahi i te aho kitea pēnei i te kikorangi metilēna, engari kāore he paenga paerewa e rite ana ki ngā whakaritenga SYBR. |
Mā te whakahaere papamuri e whakatau mēnā ka whai hua te tohu āhua ngoikore. He tino kaha te tae o te waikano here-kore, nō reira ka puta te rerekētanga mārama mā te tango i te waikano here-kore me te kore e unu i te waikano kua herea. He uaua te whakanui tairongo mā te whakapiki kukūtanga waikano. Ko te hua matua he whakapiki i te wā tango waikano. Whakahaerehia he raupapa waimehatanga mā taua tīre, taua kiriuhi, me taua kāmera tonu, ā, whakamahia anake te awhe e noho ōwehenga ana te kiato whakahiato ki te rahinga whakauru. Ka tautoko te kikorangi metilēna i te whakaritenga utanga āhua ine. Ehara i te whakamātautau ine whai awhe whānui, nā te mea ka rerekē te kaha pupuri o te kiriuhi, te tōtika o te whakawhiti, te waiwai ki te mōrahi, me te tango waikano i ia ara.
Ngā paenga hototahi: ngā mea ka taea tonu i muri i te whakawaikano
Kua whakaaturia te whakapūruatanga i muri i te whakawaikano kikorangi. Nā Flores me ōna hoa i tapahi ngā pae agarose kua whakawaikanotia, i whakahoki te DNA, i honohono, i whakawhiti hoki ki ngā pūtau. I pūrongotia he pai ake te whakapūruatanga i tērā i muri i te ethidium bromide me te UV. Ko te take pea he iti ake te tūkinotanga aho, ehara i te painga nō te waikano ake. Kia poto te whakamārama ki te rama mā, ā, whakapurengia te DNA kua whakahokia mā tētahi tukanga tango DNA i te tīre noa.
He iti ake ngā taunakitanga mō te PCR me te whakaraupapa. Ka here te toenga waikano ki ngā waikawa karihi, nō reira tangohia te waikano me te whakapurenga i mua i ngā mahi whākōkī. E tūmanakohia ana ka whai hua te DNA kua tika te whakapurenga mai i te tīre kikorangi, engari kāore anō kia tino inea te manawanui ki te waikano ka kawea tika atu mō ngā polymerase me ngā tukanga matū whakaraupapa rerekē. Kaua e whakapae noa.
He hototahi te whakahononga blot ina tino ū te raupapa: whakawhiti, whakamau, whakawaikano, tuhi pūkete, tango waikano, whakahononga. Ko taua raupapa te hoahoa a Herrin rāua ko Schmidt. He tikanga kē te whakawaikano i mua i te whakawhiti, te whakarere rānei i te tango waikano, ā, me whakamana motuhake. I muri i te tango waikano, ka haere noa ngā tikanga paerewa mō te tango pūoko me te whakamātautau anō ki te pūoko. Mō te whakawaikano taumata pūtau mā taua whānau waikano anō, me te take kāore te arorau o te paparanga kua whakamaua e hāngai ki ngā tikanga pūtau ora, tirohia te mahere whakawaikano mō te karu whakarahi.
Ka whai hua te tapahi pae mō te agarose hei whakamahi anō, me ngā oligo PAGE i kitea mā te kikorangi, mēnā ka karohia te tikanga whakamau pūmau. He whaitake ā-matū te tango mā te hiko i muri i te tango waikano mō tētahi matū here e taea ana te wetewete, engari he iti ngā raraunga rahinga hua kua whakahaeretia e hāngai pū ana ki te kikorangi metilēna. Tangohia te tote, whakapūngia rānei, i muri i te tango.
Ngā raruraru ka hoki mai anō
Ko te tikanga, ka tohu ngā pae tīre kōmā he iti rawa te papatipu mō tētahi waikano ine-tae, otirā mō ngā whāinga aho-kotahi poto, RNA rānei. Whakapikihia te utanga, whakamahia he waikano hou, whakaroatia te whakawaikano i mua i te whakapiki kukūtanga, ā, whakamutua wawe ake te tango waikano. Ko te tīre kikorangi katoa kāore he rerekētanga mārama e tohu ana i te nuinga o te wā i te nui rawa o te whakawaikano, i te iti rawa rānei o ngā huringa wai. Ko te memehatanga o ngā pae PAGE he matū e tūmanakohia ana, ehara i te whakawaikano rahua. Hopukina wawetia he whakaahua. Ko te papamuri kiriuhi pāhiwihiwi e tohu ana ki ngā kopinga, ngā mirumiru, ngā tapumati, te rūrū pāhiwihiwi, te whakawhiti wāhi-kore rānei. Kaua e whakamāoritia tērā hei utanga rerekē ki te kore he mana arowhai whakahononga. Ka tohu pea te tauira ribosome ngoikore, kua pania whānuitia, i te tūkinotanga, engari tērā pea he whakawhiti kāore i oti, he iti rawa te RNA katoa (he torutoru ki te tekau maha micrograms te utanga o ngā ara noa), he nui rawa rānei te tango waikano. Ka whakaatu te waikano i te RNA ribosome nui. Kāore e whakaatu i tētahi tāruatanga RNA motuhake. Nā te pūoko taua mahi.
Te haumaru me te whakahaere me te kore e whakanuia ngā kōrero
He māmā ake te whakahaere i te kikorangi metilēna i te ethidium bromide, engari ehara i te mea kāore he kino. E whakarōpū ana tētahi pepa raraunga haumaru o nāianei a MilliporeSigma i te waikano hei tāoke ā-waha ohorere Kāwai 4, he kino ki te horomia, me te tuku para mā tētahi ara kua whakaaetia, e ai ki te SDS kikorangi metilēna a Sigma-Aldrich. He kaha ake ngā whakatūpato tāoke ira mō te ethidium bromide, tae atu ki ngā pānga ki ngā pūtau whakaputa uri e whakapaetia ana i roto i tōna SDS ethidium bromide a Sigma-Aldrich. Ka wehea hoki ōna tīre me ōna wairewa hei para matū mōrearea i te nuinga o te wā, e ai ki te aratohu waikano waikawa karihi a Thermo Fisher. Whāia ngā SDS me ngā ture o tō whare mō ēnei mea e rua. Puritia te waikano maroke kia kati pū, kia maroke hoki; whakamahia anō te waikano mahi mēnā anake e whakaaetia ana e te kawa; rokirokitia ngā kiriuhi kua whakawaikanotia kia maroke i waenga i ngā pepa tātari; ā, hopukina ngā whakaahua tīre i mua i te mararatanga atu o ngā pae.
Kia maumahara ki te rerenga mō te pae mahi. Ka whakamātautau ngā tīre i te āheinga kia kitea i te utanga nui i raro i te rama mā. Ka whakamātautau ngā kiriuhi i te whakawhiti me te utanga i mua i te mahi tairongo a te pūoko. Kotahi te waikano mō ēnei mahi e rua, me te tairongo āhua iti, te here e taea ana te wetewete, me te raupapa mahi tino pū.