Mouse Genotyping Kit

Inombolo yekati: HCR2021A

Le mveliso yikiti eyenzelwe ukuchongwa ngokukhawuleza kwe-mouse genotypes, kubandakanywa i-DNA crude extraction kunye ne-PCR inkqubo yokukhulisa.Imveliso ingasetyenziselwa ukukhulisa i-PCR ngokuthe ngqo ukusuka kumsila wempuku, indlebe, inzwane kunye nezinye izihlunu emva kokuqhekeka okulula nguLysis Buffer kunye neProteinase k.Akukho ukugaya ebusuku, i-phenol-chloroform extraction okanye ikholomu yokucoca, elula kwaye inciphisa ixesha lokusebenzisa imifuniselo.Imveliso ifanelekile ukukhulisa amaqhekeza ekujoliswe kuwo ukuya kwi-2kb kunye neempendulo ze-PCR ezininzi kunye ne-3 pair of primers.I-2 × Mouse Tissue Direct PCR Mix iqulethe i-DNA polymerase eyenziwe ngofuzo, iMg²⁺, i-dNTPs kunye nenkqubo ye-buffer ephuculweyo yokubonelela ngobuchule obuphezulu be-amplification kunye nokunyamezela kwe-inhibitor, ukwenzela ukuba ukuphendula kwe-PCR kunokwenziwa ngokungeza i-template kunye ne-primers kunye nokubuyisela imveliso kwi-1 ×.Imveliso ye-PCR eyandisiweyo kunye nale mveliso inesiseko esivelele "A" ekupheleni kwe-3 kwaye ingasetyenziselwa ngokuthe ngqo kwi-TA cloning emva kokucocwa.

Amacandelo

Iimeko zokuGcina

Iimveliso kufuneka zigcinwe kwi -25 ~ -15 ℃ iminyaka emi-2.Emva kokunyibilika, i-Lysis Buffer inokugcinwa kwi-2 ~ 8 ℃ ukwenzela ukusetyenziswa kwexesha elifutshane, kwaye udibanise kakuhle xa usebenzisa.

Isicelo

Le mveliso ifanelekile kuhlalutyo lwe-mouse knockout, ukuxilongwa kwe-transgenic, i-genotyping njalo njalo.

Iimbonakalo

1.Ukusebenza okulula: akukho mfuneko yokukhupha i-DNA ye-genomic;

2.Usetyenziso olubanzi: lufanelekile ukukhulisa ngokuthe ngqo izicubu ezahlukeneyo zemouse.

Imiyalelo

1.Ukukhutshwa kwe-DNA ye-genomic

1) Ukulungiswa kwe-lysate

I-lysate yezicubu ilungiswa ngokwenani leesampulu zemouse eziza kuhlanjululwa (i-lysate yezicubu kufuneka ilungiswe kwindawo ngokwedosi kwaye ixutywe ngokucokisekileyo ukuze isetyenziswe), kwaye umlinganiselo we-reagents ofunekayo kwisampuli enye ngolu hlobo lulandelayo:

2) Ukulungiselela iSampuli kunye noLysis

Ukusetyenziswa kweTishu eCetyisiweyo

Thatha ubungakanani obufanelekileyo beesampulu zeethishu zempuku kwiityhubhu ze-centrifuge ezicocekileyo, yongeza i-200μL ye-lysate ye-tissue entsha kwityhubhu nganye ye-centrifuge, i-vortex kunye nokugubha, emva koko ufukamele kwi-55 ℃ ye-30mins, kwaye emva koko ushushu kwi-98℃ ye-3mins.

3) I-Centrifugation

Gubha i-lysate kakuhle kunye ne-centrifuge kwi-12,000 rpm ye-5mins.I-supernatant ingasetyenziswa njenge template yokukhulisa iPCR.Ukuba ithempleyithi iyadingeka ukugcinwa, tshintshela i-supernatant kwenye ityhubhu ye-centrifuge eyinyumba kwaye uyigcine ku -20℃ kangangeeveki ezi-2.

2.PCR Ukwandiswa

Susa i-2×Mouse Tissue Direct PCR Mix ukusuka -20℃ kwaye unyibilike emkhenkceni, xuba phezulu kwaye ulungiselele inkqubo ye-PCR yokusabela ngokwetheyibhile ilandelayo (sebenza emkhenkceni):

Phawula:

a) Imali eyongeziweyo akufanele idlule i-1/10 yenkqubo, kwaye ukuba ininzi kakhulu yongezwa, ukukhulisa i-PCR kunokuvinjelwa.

Iimeko zePCR ezicetyiswayo

Phawula:

a) Ukushisa kwe-Annealing: Ngokubhekiselele kwixabiso le-Tm le-primer, kucetyiswa ukuba usete iqondo lokushisa lokungena kwixabiso elincinci le-Tm le-primer +3 ~ 5℃.

Iingxaki eziqhelekileyo kunye nezisombululo

1.Akukho micu ekujoliswe kuyo

1) Imveliso ye-lysis egqithisileyo.Khetha elona nani lifanelekileyo letemplate, eliqhele ukuba lingabi ngaphezu kwe-1/10 yenkqubo;

2) Ubungakanani besampulu enkulu kakhulu.Nciphisa i-lysate amaxesha angama-10 kwaye emva koko ukhulise, okanye unciphise ubungakanani besampulu kunye nokuhlaziya kwakhona;

3) Iisampulu zezicubu azintsha.Kucetyiswa ukuba kusetyenziswe iisampulu zezicubu ezintsha;

4) Umgangatho ophantsi we-primer.Sebenzisa i-DNA ye-genomic yokwandisa ukuqinisekisa umgangatho we-primer kunye nokwandisa uyilo lwe-primer.

2.Ukwandisa okungangqalanga

I-1) Ubushushu be-annealing buphantsi kakhulu kwaye inombolo yomjikelezo iphezulu kakhulu.Ukwandisa ubushushu be-annealing kunye nokunciphisa inani lemijikelo;

2) Ugxininiso lwetemplate luphezulu kakhulu.Ukunciphisa inani le-template okanye uhlambulule i-template ngamaxesha angama-10 emva kokukhulisa;

3) Ubuncinci be-primer ethile.Lungiselela uyilo lokuqala.

Amanqaku

1.Ukuze ugweme ukungcoliswa komnqamlezo phakathi kweesampuli, izixhobo ezininzi zeesampuli kufuneka zilungiswe, kwaye umphezulu wezixhobo unokucocwa nge-2% yesisombululo se-sodium hypochlorite okanye i-nucleic acid cleaner emva kwesampuli nganye ukuba ukusetyenziswa ngokuphindaphindiweyo kuyadingeka.

2.Kucetyiswa ukuba kusetyenziswe izicubu ezitsha zemouse, kwaye umthamo wesampulu akufanele ube mkhulu kakhulu ukuphepha ukuchaphazela iziphumo zokukhulisa.

3.I-Lysis Buffer kufuneka inqande ukunyibilika rhoqo, kwaye inokugcinwa kwi-2~8℃ ukuze isetyenziswe ixesha elifutshane.Ukuba igcinwe kwiqondo lokushisa eliphantsi, imvula ingenzeka, kwaye kufuneka inyibilikiswe ngokupheleleyo phambi kokusetyenziswa.

4.I-PCR Mix kufuneka inqande ukunyibilika rhoqo, kwaye inokugcinwa kwi-4℃ ukuze isetyenziswe ngokuphindaphindiweyo ixesha elifutshane.

5.Le mveliso kuphela yophando lovavanyo lwesayensi kwaye akufanele isetyenziswe kuxilongo lwezonyango okanye unyango.