I-Animal Tissue Direct PCR kit-UNG (ngaphandle Kwamathuluzi Okusampula) Iphrothokholi
Incazelo yekhithi:
Sebenzisa ngokuqondile izicubu zezilwane i-lysate njengesifanekiso sokukhulisa i-PCR, ngaphandle kokukhipha okuhlukile kwe-DNA, ukuzwela okusheshayo nokuphezulu.Kwengezwe uhlelo lwe-UNG lokulwa nokunukubezeka ukuze kuqedwe imibono engamanga.
◮Akukho ukuhlanzwa kwe-DNA okuthatha isikhathi nokubizayo okudingekayo.
◮Isidingo sesampula sincane, thatha nje imbewu eyodwa.
◮Azikho izindlela zokwelapha ezikhethekile ezinjengokugaya nokuchoboza okudingekayo, futhi ukuhlinzwa kulula.
◮Isistimu ye-PCR ethuthukisiwe yenza i-PCR ibe nokucaciswa okuphezulu nokubekezelela okunamandla kuma-PCR reaction inhibitors.
Izincazelo
Le khithi isebenzisa isistimu ye-lysis buffer eyingqayizivele ukuze ikhulule ngokushesha i-DNA ye-genomic kumasampula ezicubu zezilwane ukuze kutholwe ukusabela kwe-PCR, ngakho ifaneleka ngokukhethekile ukuhlolwa kofuzo okukhulu.
Inqubo yokukhipha i-DNA ye-genomic ku-lysis buffer iqedwa phakathi kwemizuzu eyi-10-30 ku-65°C.Azikho ezinye izinqubo ezidingekayo njengokususwa kwamaprotheni kanye ne-RNA, futhi i-trace DNA ekhishiwe ingasetshenziswa njengesifanekiso sokusabela kwe-PCR.
2×PCR KululaTMI-Mix (UNG) isebenzisa i-dUTP esikhundleni se-dTTP ngesisekelo se-2×PCR EasyTMHlanganisa, bese wengeza i-UNG enzyme (Uracil-N-glycosylase) engehlisa isithunzi isifanekiso esiqukethe i-dUTP ngesikhathi esifanayo.Ngaphambi kokusabela kwe-PCR, i-enzyme ye-UNG isetshenziselwa ukuthunaza umkhiqizo we-PCR oqukethe i-uracil.I-enzyme ye-UNG ngeke ibe namuphi umthelela kusifanekiso esingaqukethe i-uracil, ngaleyo ndlela iqinisekise ukucaciswa nokunemba kokukhulisa futhi ivimbele ukuthi kungenzeka ukungcoliswa kwemikhiqizo ye-PCR ngesikhathi sokuhlolwa kofuzo okukhulu.
I-D-Taq DNA polymerase iyi-polymerase ye-DNA ethuthukiswe ngokukhethekile yi-Foregene ukuze kutholakale ukusabela okuqondile kwe-PCR.I-D-Taq DNA polymerase inokubekezelela okunamandla ezinhlobonhlobo ze-PCR reaction inhibitors, futhi ingakhulisa kahle amanani omkhondo we-DNA ezinhlelweni ezihlukene zokusabela eziyinkimbinkimbi, futhi isivinini sokukhulisa singafinyelela ku-2Kb/min, okulungele ikakhulukazi ukusabela kwe-Direct PCR.
Izingxenye zekhithi
| Ingxenye I | Isivimbeli AL |
| I-Foregene Protease | |
| I-6× I-DNA Yokulayisha Ibhafa | |
| Ingxenye II | 2×PCR KululaTMImiksi(UNG) |
Izici&izinzuzo
■Akukho ukuhlanzwa kwe-DNA okuchitha isikhathi nokubizayo
∎ Isidingo sesampula sincane, njengoba izicubu zezilwane ezingama-5mg zingahlolwa.
■ Akukho ukwelashwa okukhethekile okufana nokugaya nokuchoboza okudingekayo, futhi ukuhlinzwa kulula.
■ Uhlelo lwe-PCR olulungiselelwe lenza i-PCR ibe nokucaciswa okuphezulu nokubekezelela okuqinile kuma-PCR reaction inhibitors.
■ Uhlelo lwe-PCR lokulwa nokunukubezeka 2×I-PCR elulaTMI-Mix (UNG) iqeda ngempumelelo ukungcola okubangelwa imikhiqizo ye-PCR futhi iqinisekisa ukucaciswa nokunemba kokukhulisa.
Imingcele yekhithi
Isicelo: izicubu ezihlukahlukene zezilwane.
I-DNA ekhishwe kusampula ye-lysis: isetshenziswa kuphela njengesifanekiso se-PCR.
Ikhithi ingasetshenziselwa lezi zinhloso ezilandelayo: ukuhlonza i-transgenosis, i-genotyping yezilwane, njll.
Ukugeleza komsebenzi
Isitoreji kanye Nempilo yeshelufu
Ingxenye I yale kit kufanele igcinwe ku-2-8℃.
I-reagent Buffer AL ingagcinwa izinyanga eziyi-12 ngaphansi kwezimo ezomile;ingagcinwa ku-2-8℃ukuze ugcine isikhathi eside.
I-Reagent Foregene Protease inefomula eyingqayizivele.Ukuqinisekisa umsebenzi wayo nokuzinza, sicela uyigcine ku-4℃izinyanga ezingu-12.
I-Reagent 6×I-DNA Loading Buffer ingagcinwa ku-4℃noma -20℃isikhathi eside.
Ingxenye II yale kit kufanele igcinwe ku -20℃.
I-Reagent 2×I-PCR elulaTMI-Mix(UNG), uma isetshenziswa njalo, ingabuye igcinwe ku-4℃ukugcina isikhathi esifushane (sebenzisa phakathi kwezinsuku eziyi-10).
