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Ikhithi ye-PCR ye-Animal Tissue Direct (ngaphandle Kwamathuluzi Okusampula) Iphrothokholi

Incazelo yekhithi:

Sebenzisa ngokuqondile izicubu zezilwane i-lysate njengesifanekiso sokukhulisa i-PCR, ngaphandle kokukhipha okuhlukile kwe-DNA, ukuzwela okusheshayo nokuphezulu.

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.

amandla angaphambili


Imininingwane Yomkhiqizo

Omaka bomkhiqizo

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 inokubekezelela okuqinile kwe-PCR reaction inhibitors, futhi ingasebenzisa i-lysate yesampula ukuze ihlolwe njengesifanekiso sokukhulisa kahle nokuqondile.Lesi sikhungo siqukethe i-Foregene D-Taq DNAPolymerase, i-dNTPs, i-MgCl2, isigcinalwazi sokusabela, i-PCR optimizer nesiqinisi.

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.

Imininingwane

50×20µl rxns, 200×20µl rxns, 500×20µl rxns, 2000×20µl rxns

Izingxenye zekhithi

Ingxenye I

Isivimbeli AL

I-Foregene Protease

I-6× I-DNA Yokulayisha Ibhafa

Ingxenye II

2× PCR KululaTMHlanganisa

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.

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

i-wk flw-animal tissue direct PCR

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-2-8ukuze ugcine isikhathi eside.

 I-Reagent Foregene Protease inefomula eyingqayizivele.Ukuqinisekisa umsebenzi wayo nokuzinza, sicela uyigcine ku-4izinyanga ezingu-12.

 I-Reagent 6×I-DNA Loading Buffer ingagcinwa ku-4noma -20isikhathi eside.

Ingxenye II yale kit kufanele igcinwe ku -20.

 I-Reagent 2×I-PCR elulaTMImiksi ingagcinwa izinyanga eziyi-12 ku -20;uma isetshenziswa njalo, ingabuye igcinwe ku-4ukugcina isikhathi esifushane (sebenzisa phakathi kwezinsuku eziyi-10).


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