I-Zebra Fish Direct PCR Kit-UNG Direct PCR Lysis Reagent(i-zebrafish)
Izincazelo
Le kit isebenzisa isistimu ye-lysis buffer eyingqayizivele ukuze ikhulule ngokushesha i-DNA ye-genomic ku-zebrafish nezinye izicubu zezinhlanzi zasemanzini ahlanzekile, amaphiko omsila noma amasampula amaqanda ezinhlanzi zokusabela 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.
I-2× PCR EasyTM Mix (UNG) isebenzisa i-dUTP esikhundleni se-dTTP ngesisekelo se-2× PCR EasyTM Mix, futhi yengeza i-UNG enzyme (Uracil-N-glycosylase) engalulaza ithempulethi equkethe 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 nokuvimbela amathuba okuhlolwa kofuzo okukhulu.Ukungcoliswa kwemikhiqizo ye-PCR kwenzekile.
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 | I-Buffer FP |
I-Foregene Protease | |
I-6× I-DNA Yokulayisha Ibhafa | |
Ingxenye II | 2×PCR KululaTMImiksi(UNG) |
Iziyalezo |
Izici&izinzuzo
■ Akukho ukuhlanzwa kwe-DNA okuthatha isikhathi nokubizayo okudingekayo.
■ Isidingo sesampula sincane, njengoba nje kungahlolwa i-fish caudal fin engu-1mg noma amaqanda ayi-10 ezinhlanzi.
■ Azikho izindlela zokwelapha ezikhethekile ezinjengokugaya nokuchoboza okudingekayo, futhi ukuhlinzwa kulula.
■ Uhlelo lwe-PCR olulwa nokungcola 2× PCR EasyTM Mix (UNG), olungaqeda ngempumelelo ukungcola okubangelwa imikhiqizo ye-PCR futhi luqinisekise ukunemba nokunemba kokukhulisa.
■ Uhlelo lwe-PCR olulungiselelwe lenza i-PCR ibe nokucaciswa okuphezulu nokubekezelela okuqinile kuma-PCR reaction inhibitors.
Imingcele yekhithi
Isicelo: Izinhlanzi ze-Zebra nezinye izinhlanzi zamanzi ahlanzekile.
I-DNA ekhishwe kusampula ye-lysis: isetshenziswa kuphela njengesifanekiso se-PCR.
Ikhithi ingasetshenziselwa lezi zinhloso ezilandelayo: ukuhlonza i-transgenosis, i-genotyping yezilwane, njll.
Ukuhamba komsebenzi
Isitoreji kanye Nempilo yeshelufu
Ingxenye I yale kit kufanele igcinwe ku-2-8℃.
I-reagent Buffer FP ingagcinwa izinyanga eziyi-12 ngaphansi kwezimo ezomile;ingagcinwa ku-2-8℃ ukuze igcinwe isikhathi eside.
v I-Reagent Forgene Protease inefomula eyingqayizivele.Ukuqinisekisa ukusebenza kwayo nokuzinza, sicela uyigcine ku-4℃ izinyanga eziyi-12.
I-vReagent 6×DNA Loading Buffer ingagcinwa ku-4℃ noma -20℃ isikhathi eside.
Ingxenye II yale khithi kufanele igcinwe ku -20 ℃.
I-vReagent 2×PCR EasyTM Mix(UNG), uma isetshenziswa njalo, ingabuye igcinwe ku-4℃ ukuze igcinwe isikhathi esifushane (isetshenziswe ngaphakathi kwezinsuku eziyi-10).