I-PCR FAQ 1. I-negethivu yamanga, alikho ibhande lokukhulisa elivelayo 2. Ukuphozithiza okungelona iqiniso 3. Amabhande okukhulisa amagama angaqondile avela 4. Kuvela amapheshana adonsayo noma ama-smear strips:

1Inegethivu yamanga, alikho ibhendi ekhulisiwe evelayo Izici ezibalulekile zokusabela kwe-PCR yilezi

① ukulungiswa kwesifanekiso se-nucleic acid

② ikhwalithi yokuqala kanye nokucacisa

③ ikhwalithi ye-enzyme kanye

④ Izimo zomjikelezo we-PCR. Ukuthola izizathu, ukuhlaziya kanye nocwaningo kufanele futhi kwenziwe kulezi zixhumanisi ezingenhla.

Isifanekiso:

① Isifanekiso siqukethe amaprotheni angcolile

② Isifanekiso siqukethe i-Taq enzyme inhibitors

③ Amaprotheni akusifanekiso awagaywe futhi asusiwe, ikakhulukazi ama-histones kuma-chromosome

④ Kulahleke okuningi kakhulu ngesikhathi kukhishwa futhi kulungiswa isifanekiso, noma i-phenol ihogelwe

⑤Isifanekiso se-nucleic acid asishintshiwe ngokuphelele. Uma izinga lama-enzyme nama-primer lilihle, uma ama-amplification bands engabonakali, kungenzeka ukuthi kukhona okungalungile ngenqubo yokugaya ye-specimen noma inqubo yokukhipha i-nucleic acid yesifanekiso. Ngakho-ke, isisombululo sokugaya esisebenzayo nesizinzile kufanele silungiswe, futhi inqubo kufanele ilungiswe futhi akufanele ishintshwe ngokuthanda kwakho. . Ukungasebenzi kwe-enzyme: Kuyadingeka ukufaka esikhundleni se-enzyme entsha, noma usebenzise womabili ama-enzyme amadala namasha ngesikhathi esisodwa ukuze uhlaziye ukuthi ama-negetive angamanga abangelwa ukulahleka noma umsebenzi onganele we-enzyme. Kufanele kuqashelwe ukuthi ngezinye izikhathi i-enzyme ye-Taq iyakhohlwa.Ama-Primers: Ikhwalithi ye-Primer, ukugxilwa kwe-primer, nokuthi ukugxila kwe-primer ezimbili kuyalingana yizizathu ezivamile zokuhluleka kwe-PCR noma amabhande okukhulisa angagculisi kanye nokusabalalisa kalula. Kunezinkinga ngekhwalithi ye-primer synthesis kwamanye amaqoqo. Enye yalezi ziqalo ezimbili inokugxila okuphezulu kanti enye ine-concentration ephansi, okuholela ekukhuliseni okuphansi kwe-asymmetric.

Izinyathelo zokuphikisa yilezi:

① Khetha iyunithi enhle yokuqala.

② Ukugxila kwama-primers akufanele kubheke inani le-OD kuphela, kodwa futhi kunake isisombululo se-primer stock se-agarose gel electrophoresis. Kufanele kube namabhendi okuqala, futhi ukukhanya kwamabhendi amabili okuqala kufanele kufane. Isibonelo, i-primer eyodwa inebhendi kanti enye i-primer ayinalo ibhande. Emapheshana, i-PCR ingase yehluleke ngalesi sikhathi futhi kufanele ixazululwe ngokuxoxisana neyunithi yokuhlanganisa yokuqala. Uma i-primer eyodwa inokukhanya okuphezulu kanti enye inokukhanya okuphansi, bhalansisa ukugxilisa ingqondo lapho uhlanza ama-primer.

③ Ama-Primers kufanele agcinwe ekugxiliseni okuphezulu kanye nenani elincane ukuvimbela ukuqhwa okuphindaphindiwe nokuncibilika noma ukugcinwa isikhathi eside esiqandisini, okungase kubangele ukuwohloka nokuwohloka kweziqalo.

④Idizayini ye-primer ayinangqondo, njengokuthi ubude be-primer abanele, ama-dimers akhiwa phakathi kwama-primer, njll. Ukugxiliswa kwe-Mg2+: Ukugxiliswa kwe-ion ye-Mg2+ kunomthelela omkhulu ekusebenzeni kahle kokukhulisa i-PCR. Uma ukugxila kuphezulu kakhulu, kunganciphisa ukucaciswa kokukhulisa i-PCR. Uma ukugxilisa ingqondo kuphansi kakhulu, kuzothinta isivuno sokukhulisa i-PCR futhi kubangele nokukhulisa i-PCR kuhluleke ngaphandle kwamabhendi okukhulisa. Izinguquko kuvolumu yokusabela: Ngokuvamile amavolumu asetshenziselwa ukukhulisa i-PCR ama-20ul, 30ul, kanye nama-50ul. Noma i-100ul, ukuthi iyiphi ivolumu okufanele isetshenziselwe ukukhulisa i-PCR isethwe ngokwezinjongo ezihlukene zocwaningo lwesayensi nokuhlolwa komtholampilo. Ngemuva kokwenza ivolumu encane, njenge-20ul, bese wenza ivolumu enkulu, kufanele ulandele izimo, ngaphandle kwalokho izohluleka kalula. Izizathu zomzimba: I-Denaturation ibaluleke kakhulu ekukhuliseni i-PCR. Uma izinga lokushisa le-denaturation liphansi futhi isikhathi se-denaturation sisifushane, ama-negative amanga cishe angenzeka; izinga lokushisa le-annealing liphansi kakhulu, elingabangela ukukhulisa okungaqondile futhi kunciphise ukusebenza kahle kokukhulisa i-amplification. Izinga lokushisa le-anneal liphezulu kakhulu. Kuthinta kakhulu ukuboshelwa kweziqalo ezifanekisweni futhi kunciphisa ukusebenza kahle kokukhulisa i-PCR. Kwesinye isikhathi kuyadingeka ukusebenzisa ithemometha evamile ukuze uhlole izinga lokushisa elincibilikayo, i-annealing kanye nesandiso ku-amplifier noma ebhodweni elincibilika emanzini. Lesi futhi esinye sezizathu zokwehluleka kwe-PCR. Ukuhluka kokulandelanisa okuqondiwe: Uma ukulandelana okuqondiwe kuguqulwa noma kususwa, okuthinta ukubophezela okuqondile kwe-primer kusifanekiso, noma isiqalo nesifanekiso silahlekelwa ukulandelana okuhambisanayo ngenxa yokususwa kwesegimenti ethile yokulandelana okuqondiwe, ukukhulisa i-PCR. ngeke aphumelele.

2.okungelona iqiniso Ibhendi yokukhulisa i-PCR evelayo ihambisana nebhendi yokulandelana okuqondiwe, futhi ngezinye izikhathi ibhendi iyahleleka kakhulu futhi igqame. Idizayini ye-primer engafanele: Ukulandelana kokukhulisa okukhethiwe kune-homology nokulandelana kokukhulisa okungahlosiwe, ngakho-ke lapho kwenziwa ukukhulisa i-PCR, umkhiqizo we-PCR okhulisiwe uwukulandelana okungahlosiwe. Uma ukulandelana okuqondiwe kukufishane kakhulu noma i-primer imfushane kakhulu, amaphozithivu angamanga angase avele kalula. Ama-primers adinga ukuklanywa kabusha. Ukungcoliswa okuphambanayo kokulandelana okuqondiwe noma imikhiqizo yokukhulisa amandla: Kunezizathu ezimbili zalokhu kungcola: Esokuqala, ukungcoliswa okuphambanayo kwayo yonke i-genome noma izingcezu ezinkulu, okuholela ekutholeni amanga. Lokhu okungelona iqiniso kungaxazululwa ngalezi zindlela ezilandelayo: Qaphela futhi ube mnene lapho usebenza ukuze uvimbele ukulandelana okuqondiwe ukuthi kungamuncwa kusibhamu esiyisampula noma kukhishwe ishubhu le-centrifuge. Ngaphandle kwama-enzyme nezinto ezingakwazi ukumelana namazinga okushisa aphezulu, wonke ama-reagents noma izinto zokusebenza kufanele zibulawe inzalo ngokucindezela okukhulu. Wonke amashubhu e-centrifuge kanye namathiphu e-pipette yomjovo wesampula kufanele asetshenziswe kanye. Uma kunesidingo, amashubhu okusabela kanye nama-reagents akhanyisa ukukhanya kwe-ultraviolet ngaphambi kokwengeza isifanekiso sokubhubhisa ama-nucleic acid akhona. Okwesibili ukungcoliswa kwezingcezu ezincane zama-nucleic acid emoyeni. Lezi zingcezu ezincane zifushane kunokulandelana okuqondiwe, kodwa zine-homology ethile. Angakwazi ukuhlukaniswa komunye nomunye, futhi ngemva kokuhambisana nama-primers, imikhiqizo ye-PCR ingakhuliswa, okuholela emiphumeleni engamanga, engancishiswa noma iqedwe ngezindlela ze-PCR ezifakwe isidleke.

 

3.Amabhendi angacaciswanga okukhulisa izwi ayavela Amabhendi avela ngemva kokukhulisa i-PCR awahambisani nosayizi olindelekile, omkhulu noma omncane, noma womabili amabhendi okukhulisa izwi kanye namabhande angacacisiwe wokukhulisa avela ngesikhathi esisodwa. Izizathu zokuvela kwamabhendi angacacisiwe yilezi: okokuqala, ama-primers awahambisani ngokuphelele nokulandelana okuqondiwe, noma ama-primer ahlanganisa ukwenza ama-dimers. Isizathu sesibili ukuthi i-Mg2+ ion concentration iphezulu kakhulu, izinga lokushisa le-anneal liphansi kakhulu, futhi inani lemijikelezo ye-PCR liphezulu kakhulu. Isici sesibili izinga kanye nenani le-enzyme. Ama-enzyme avela kweminye imithombo avame ukuthambekela kumabhande angaqondile kodwa ama-enzyme akweminye imithombo awakwenzi. Amanani amaningi ama-enzyme ngezinye izikhathi angaholela ekukhuliseni okungaqondile. Izinyathelo zokuphikisa zihlanganisa: hlela kabusha ama-primers uma kunesidingo. Yehlisa inani le-enzyme noma ubeke omunye umthombo. Yehlisa inani lama-primer, wandise inani lesifanekiso ngokufanele, futhi unciphise inani lemijikelezo. Khulisa izinga lokushisa le-anneal ngokufanele noma sebenzisa indlela yamaphoyinti okushisa amabili (i-denaturation ku-93°C, i-annealing kanye nesandiso cishe ngo-65°C).

 

4.Ukuhudula okuxekethile noma ama-smears avela Ukukhulisa i-PCR ngezinye izikhathi kubonakala njengamabhande agcotshiwe, amabhande afana neshidi noma amabhande afana nokhaphethi. Izizathu zivame ukubangelwa i-enzyme eningi noma ikhwalithi engeyinhle ye-enzyme, ukugxiliswa kwe-dNTP okuphezulu kakhulu, ukugxila kwe-Mg2+ ephezulu kakhulu, izinga lokushisa eliphansi kakhulu le-anneal, kanye nemijikelezo eminingi kakhulu. Izinyathelo zokulwa zifaka: ① Yehlisa inani le-enzyme, noma shintsha i-enzyme ngomunye umthombo. ②Yehlisa ukugcwala kwe-dNTP. Yehlisa ngokufanelekile ukugxila kwe-Mg2+. Khulisa inani lezifanekiso futhi unciphise inani lemijikelezo