Ikhithi ye-Mouse Genotyping

Inombolo yekati: HCR2021A

Lo mkhiqizo uyikhithi eklanyelwe ukuhlonza ngokushesha ama-mouse genotypes, okuhlanganisa i-DNA crude extraction kanye nesistimu yokukhulisa i-PCR.Umkhiqizo ungasetshenziselwa ukukhulisa i-PCR ngokuqondile kusukela kumsila wegundane, indlebe, uzwane nezinye izicubu ngemva kokuqhekeka okulula ngu-Lysis Buffer kanye ne-Proteinase k.Akukho ukugaya ebusuku, isizinda se-phenol-chloroform noma ukuhlanzwa kwekholomu, okulula futhi okufinyeza isikhathi sokuhlolwa.Umkhiqizo ulungele ukukhuliswa kwezingcezu eziqondiwe kuze kufike ku-2kb nokusabela kwe-multiplex PCR kuze kufike kumapheya angu-3 weziqalo.I-2×Mouse Tissue Direct PCR Mix iqukethe i-DNA polymerase eyenziwe ngofuzo, i-Mg.²⁺, ama-dNTP kanye nesistimu ye-buffer elungiselelwe ukunikeza ukusebenza kahle kokukhulisa ukuphakama nokubekezelela i-inhibitor, ukuze ukusabela kwe-PCR kwenziwe ngokungeza ithempulethi nama-primer kanye nokubuyisela amanzi kumkhiqizo ku-1×.Umkhiqizo we-PCR okhuliswe ngalo mkhiqizo unesisekelo esithi “A” esivelele ekugcineni kwe-3′ futhi ungasetshenziswa ngokuqondile ekwenzeni i-TA cloning ngemva kokuhlanzwa.

Izingxenye

Izimo Zokugcina

Imikhiqizo kufanele igcinwe ku -25~-15℃ iminyaka emi-2.Ngemva kokuncibilika, i-Lysis Buffer ingagcinwa ku-2~8℃ ukuze isetshenziswe isikhathi esifushane kaningi, futhi ixube kahle uma usebenzisa.

Isicelo

Lo mkhiqizo ulungele ukuhlaziywa kwegundane, ukutholwa kwe-transgenic, i-genotyping nokunye.

Izici

1.Ukusebenza okulula: asikho isidingo sokukhipha i-DNA ye-genomic;

2.Uhlelo lokusebenza olubanzi: lulungele ukukhuliswa okuqondile kwezicubu ezihlukahlukene zegundane.

Iziyalezo

1.Ukukhishwa kwe-genomic DNA

1) Ukulungiswa kwe-lysate

I-tissue lysate ilungiswa ngokuya ngenani lamasampula egundane azokhishwa (i-lysate yezicubu kufanele ilungiswe endaweni ngokuya ngomthamo futhi ixutshwe kahle ukuze isetshenziswe), kanye nenani lama-reagents adingekayo kwisampula eyodwa limi kanje:

2) Isampula Ukulungiselela kanye Lysis

Ukusetshenziswa Kwezicubu Okunconyiwe

Thatha inani elifanele lamasampula ezicubu zegundane kumashubhu e-centrifuge ahlanzekile, engeza u-200μL we-lysate yezicubu ezintsha kushubhu ngayinye ye-centrifuge, i-vortex kanye nokunyakazisa, bese ufukamela ku-55℃ imizuzu engu-30, bese ushisa ku-98℃ imizuzu engu-3.

3) I-Centrifugation

Nyakazisa i-lysate kahle bese uyi-centrifuge ku-12,000 rpm imizuzu emi-5.I-supernatant ingasetshenziswa njengesifanekiso sokukhulisa i-PCR.Uma isifanekiso sidingeka ukuze sigcinwe, dlulisela i-supernatant kwelinye ishubhu le-centrifuge eliyinyumba futhi ugcine ku -20℃ amaviki angu-2.

2.Ukukhulisa i-PCR

Susa i-2×Mouse Tissue Direct PCR Mix kusuka ku-20℃ bese uncibilikisa eqhweni, uhlanganise ubheke phansi bese ulungisa uhlelo lokusabela lwe-PCR ngokwetafula elilandelayo (sebenzisa eqhweni):

Qaphela:

a) Inani elingeziwe akumele lidlule u-1/10 wesistimu, futhi uma kungeziwe kakhulu, ukukhulisa i-PCR kungase kuvinjelwe.

Izimo ze-PCR ezinconyiwe

Qaphela:

a) Izinga lokushisa le-annealing: Ngokubhekisela enanini le-Tm le-primer, kunconywa ukuthi usethe izinga lokushisa lokudonsa libe kunani elincane le-Tm le-primer +3~5℃.

Izinkinga Ezivamile Nezixazululo

1.Ayikho imicu eqondiwe

1) Umkhiqizo we-lysis oweqile.Khetha inani elifanele kakhulu lesifanekiso, ngokuvamile lingabi ngaphezu kwe-1/10 yesistimu;

2) Usayizi wesampula omkhulu kakhulu.Nciphisa i-lysate izikhathi ezingu-10 bese ukhulisa, noma unciphise usayizi wesampula futhi uphinde uhlaziye;

3) Amasampula ezicubu awamusha.Kunconywa ukusebenzisa amasampula ezicubu ezintsha;

4) Ikhwalithi ye-primer ephansi.Sebenzisa i-genomic DNA ukuze ukhulise ukuze uqinisekise ikhwalithi yokuqala futhi uthuthukise umklamo wokuqala.

2.Ukukhulisa okungaqondile

1) Izinga lokushisa le-anneal liphansi kakhulu futhi inombolo yomjikelezo iphezulu kakhulu.Khulisa izinga lokushisa le-annealing futhi unciphise inani lemijikelezo;

2) Ukugxiliswa kwesifanekiso kuphezulu kakhulu.Yehlisa inani lesifanekiso noma unciphise isifanekiso izikhathi eziyi-10 ngemuva kokukhulisa;

3) Ukucaciswa kwe-primer okungalungile.Lungiselela umklamo wokuqala.

Amanothi

1.Ukuze ugweme ukungcoliswa phakathi kwamasampula, amathuluzi amaningi okusampula kufanele alungiswe, futhi ingaphezulu lamathuluzi lingahlanzwa ngesisombululo se-sodium hypochlorite esingu-2% noma isicoci se-nucleic acid ngemva kwesampula ngayinye uma ukusetshenziswa okuphindaphindiwe kuyadingeka.

2.Kunconywa ukusebenzisa izicubu zegundane ezintsha, futhi umthamo wesampula akufanele ube mkhulu kakhulu ukugwema ukuthikameza imiphumela yokukhulisa.

3.I-Lysis Buffer kufanele igweme ukuncibilika okuvamisile, futhi ingagcinwa ku-2~8℃ ukuze isetshenziswe isikhathi esifushane kaningi.Uma igcinwe ezingeni lokushisa eliphansi, imvula ingase ibe khona, futhi kufanele incibilike ngokugcwele ngaphambi kokusetshenziswa.

4.I-PCR Mix kufanele igweme ukuncibilika kweqhwa njalo, futhi ingagcinwa ku-4℃ ukuze isetshenziswe ngokuphindaphindiwe isikhathi esifushane.

5.Lo mkhiqizo ngowocwaningo lokuhlola lwesayensi kuphela futhi akufanele usetshenziswe ekuxilongweni komtholampilo noma ekwelapheni.