Ikiti ye-PCR yeZilwanyana eziNgqo (ngaphandle kweZixhobo zokuSampula) iProtocol
Iinkcazelo
Le khithi isebenzisa inkqubo ye-lysis buffer ekhethekileyo ukukhulula ngokukhawuleza i-DNA ye-genomic kwiisampuli zezicubu zezilwanyana ukwenzela ukusabela kwe-PCR, ngoko ke ifaneleke ngokukodwa kuvavanyo olukhulu lwemfuzo.
Inkqubo yokukhulula i-DNA ye-genomic kwi-lysis buffer igqityiwe ngaphakathi kwe-10-30 imizuzu kwi-65 ° C.Azikho ezinye iinkqubo ezifana neprotheyini kunye nokususwa kwe-RNA ezifunekayo, kwaye umkhondo okhutshiweyo we-DNA ungasetyenziswa njenge template ye-PCR reaction.
2 × PCR EasyTMUmxube unonyamezelo oluqinileyo kwi-PCR reaction inhibitors, kwaye unokusebenzisa i-lysate yesampuli ukuba ivavanywe njengethemplate yokukhulisa ngokufanelekileyo kunye nokucacileyo.Le reagent iqulethe Foregene D-Taq DNAPolymerase, dNTPs, MgCl2, i-reaction buffer, i-PCR optimizer kunye ne-stabilizer.
I-D-Taq DNA polymerase yipolymerase ye-DNA ephuhliswe ngokukodwa yi-Foregene ukwenzela ukuphendula ngokuthe ngqo kwe-PCR.I-D-Taq DNA polymerase inonyamezelo oluqinileyo kwiintlobo ezahlukeneyo ze-PCR reaction inhibitors, kwaye inokwandisa ngokufanelekileyo izixa ze-DNA kwiinkqubo ezahlukeneyo zokusabela ezintsonkothileyo, kunye nesantya sokukhulisa sinokufikelela kwi-2Kb / min, efanelekileyo kakhulu kwi-Direct PCR reaction.
Iinkcukacha
50×20µl rxns, 200×20µl rxns, 500×20µl rxns, 2000×20µl rxns
Amacandelo ekhithi
Icandelo I | Isithinteli AL |
I-Foregene Protease | |
I-6 × i-DNA yokulayisha i-Buffer | |
Icandelo II | 2 × PCR EasyTMXuba |
Iimpawu&izinto eziluncedo
■Akukho xesha lidla kwaye libiza ukucocwa kweDNA
∎ Imfuno yesampulu incinci, kancinane njenge-5mg inyama yesilwanyana inokuvavanywa.
∎ Akukho nyango lukhethekileyo olunjengokusila nokutyumza olufunekayo, kwaye utyando lulula.
■ Inkqubo ye-PCR ephuculweyo yenza ukuba i-PCR ibe nezinto ezicacileyo eziphezulu kunye nokunyamezelana okunamandla kwii-PCR reaction inhibitors.
Iiparamitha zeKit
Isicelo: iintlobo ezahlukeneyo zezicubu zezilwanyana.
I-DNA ekhutshwe kwisampuli ye-lysis: isetyenziswe kuphela njenge-template ye-PCR.
Ikiti ingasetyenziselwa ezi njongo zilandelayo: ukuchongwa kwe-transgenosis, i-genotyping yezilwanyana, njl.
Ukuhamba komsebenzi
Ugcino kunye nobomi beShelf
Inxalenye I yale khithi kufuneka igcinwe kwi-2-8℃.
I-reagent Buffer AL inokugcinwa iinyanga ezili-12 phantsi kweemeko ezomileyo;inokugcinwa kwi-2-8℃yokugcina ixesha elide.
I-Reagent Foregene Protease inefomula eyodwa.Ukuqinisekisa umsebenzi wayo kunye nokuzinza, nceda uyigcine ku-4℃iinyanga ezili-12.
I-Reagent 6×I-DNA Loading Buffer inokugcinwa kwi-4℃okanye -20℃ixesha elide.
Inxalenye II yale khithi kufuneka igcinwe kwi -20℃.
I-Reagent 2×PCR EasyTMUmxube ungagcinwa iinyanga ezili-12 kwi -20℃;ukuba isetyenziswa rhoqo, inokugcinwa ngo-4℃ukugcinwa kwexesha elifutshane (sebenzisa phezulu kwiintsuku ezili-10).